Na+ Regulation of the Cardiac Excitation‐ Contraction‐Relaxation Coupling

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

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

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

Supported by

Contents Acknowledgements ...... 3 Supported by ...... 4 Contents ...... 5 List of abbreviations ...... 7 Papers included in this thesis ...... 9 1. Introduction ...... 10 1.1 The heart ...... 10 1.2 The excitation‐contraction‐relaxation cycle in cardiomyocytes ...... 11 1.2.1 The excitation – the action potential ...... 11 1.2.2. The contraction ‐ cytosolic Ca2+ release ...... 12 1.2.3 The relaxation – cytosolic Ca2+ removal ...... 14 1.3. Na+ as determinant of Ca2+ transients in cardiomyocytes ...... 16 1.3.1 Na+ balance in cardiomyocytes ...... 16 1.3.2 Voltage gated Na+ channels ...... 16 1.3.3 The Na+/Ca2+ exchanger ...... 16 1.3.4 The Na+/K+ ATPase ...... 17 1.4 Subcellular regulation of Na+ fluxes in cardiomyocytes ...... 19 1.4.1 The ankyrins ...... 19 1.4.2 Localized subdomains for Na+ in cardiomyocytes ...... 20 1.4.3 Do Na+ hotspots and coldspots exist in cardiomyocytes? ...... 22 1.5 Arrhythmias due to Ca2+ overload in cardiomyocytes ...... 23 1.5.1 Afterdepolarizations and Ca2+ waves ...... 23 1.5.2 Ankyrin B syndrome ...... 24 1.5.3 Ca2+ channel blockers ‐ a new treatment option for ankyrin B syndrome? ...... 24 2. Main aims ...... 26 3. Methods ...... 27 3.1 Animal models ...... 27 3.2 Isolated cardiomyocytes ...... 28 3.3 Electrophysiological methods ...... 29 3.3.1 Voltage clamp ...... 29 3.3.2 Protocol for NKA dependent regulation of NCX ...... 30 3.3.3 Methodological considerations regarding NKA dependent regulation of NCX ...... 31 3.3.4 Field‐stimulation ...... 31 3.4 Immunocytochemistry ...... 32 3.5 Detubulation ...... 32 3.6 Peptide pulldown assay ...... 33

3.7 Fluorescence microscopy ...... 34 3.8 Ca2+ imaging with confocal microscopy ...... 35 3.9 Western blot ...... 35 3.10 Computer models ...... 35 4. Summary of results ...... 37 4.1 Paper 1 ...... 37 4.2 Paper 2 ...... 37 4.3 Paper 3 ...... 38 5. Discussion ...... 39

5.1 Subcellular distribution of NKA1 and ‐2 isoforms ...... 39

5.2 NKAα2 controls NCX‐activity ...... 40 5.3 The MAB‐peptide ‐ a disruptor peptide of the NKA‐coupling to ankB ...... 41 5.4 AnkB as basis for NKA dependent regulation of NCX ...... 42 5.5 NKA‐regulation of Ca2+ fluxes through control of NCX‐activity ...... 43 5.6 Verapamil prevents Ca2+ waves in ankB+/‐ cardiomyocytes ...... 45 6. Conclusions ...... 47 7. Reference list ...... 48 8. Errata ...... 55 9. Appendix: Paper 1‐3 ...... 57

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

2+ 2+ Ca c cytosolic Ca 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

2+ ECa equilibrium potential for Ca

+ ENa equilibrium potential for Na

+ 2+ ENa/Ca equilibrium potential for Na /Ca exchange

ECG electrocardiogram

ECR‐cycle excitation‐contraction‐relaxation cycle

2+ 2+ ICaL Ca current through L‐type Ca channels

+ IKr delayed rectifier K current

+ IK1 inward rectifier K current

INCX NCX current

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

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

2+ 2+ 3) ICaL inhibition prevents arrhythmogenic Ca waves caused by abnormal Ca 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.

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.

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

2+ 2+ 2+ in cytosolic Ca concentration (Ca c), the Ca 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.

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

1.2.2. The contraction ‐ cytosolic Ca2+ release 2+ In a resting cardiomyocyte, Ca 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

2+ 2+ cell membrane and regulated influx and efflux of Ca allow rapid changes of Ca c. Together with

2+ 2+ the transient and regular changes of Ca c between diastole and systole, this makes Ca an efficient

2+ 2+ 2+ messenger [10]. The increase in Ca c comes from sarcolemmal Ca influx during the AP and Ca release from the SR.

1.2.2.1 Sarcolemmal Ca2+ influx 2+ 2+ The Ca transient is initiated by transsarcolemmal Ca 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

2+ [14‐16]. Regulation of ICaL is a main determinant of Ca 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

+ + 2+ depolarized, the cytosolic Na concentration (Na c) is high and before ICaL causes local cytosolic Ca elevation [18]. Whether Ca2+ influx via NCX can trigger SR Ca2+ release is controversial. The role of

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

2+ 2+ of Ca from the SR, which is not sufficient to produce a detectable increase in the average Ca 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,

2+ 2+ which together induce the rise in average Ca c and thus provide the Ca 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.

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.

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

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

1.3.1 Na+ balance in cardiomyocytes 2+ + Cardiac ECR‐coupling and Ca 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

2+ + + + + by secondary active transport, including Ca (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.

+ + 2+ The NCX operating mode is determined by Na c , extracellular concentration of Na and Ca and the

+ 2+ membrane potential (Em), where the equilibrium potential for Na /Ca exchange, ENa/Ca = 3ENa – 2ECa

+ 2+ (ENa and ECa are the equilibrium potentials for Na and Ca ). 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

+ + 2+ Em > ENa/Ca because of high Na c due to opening of the voltage gated Na channels and low Ca 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

17 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.

2+ Heterozygous knockout mice lacking 1 are hypo‐contractile with reduced Ca transients, whereas

2+ the heterozygous 2 knockouts are hyper‐contractile with increased Ca transients [54], coupling

2+ NKA2 to control of Ca 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

+ 2+ activity leading to increased Na ‐extrusion, thus counteracting the concomitant increase in Ca 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.

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

19 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

+ 2+ 2+ the bulk Na c. The concept of subcellular pockets is well established for Ca , where the Ca 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

2+ dyadic cleft and not the average Ca 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:

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].

 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 

+ 2+ sensed by the NCX independently of the bulk Na c and whether this controls Ca 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.

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+

2+ overload in cardiomyocytes. High Ca 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

2+ cluster of RyRs to the next along the SR membrane, thus increasing Ca 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].

2+ 2+  Depolarization of the resting Em: The released Ca ions during a Ca wave can either be pumped

into the SR by SERCA2, or be extruded by the NCX generating a inward current, Iti, due to the

+ 2+ inward movement of Na ions and thus depolarization of Em [102]. If sufficient amounts of Ca 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

24 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

2+ 2+ reducing [Ca ]c and/or SR Ca 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.

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:

+ + + 2+ 1) Study the role of the Na /K ATPase 2isoform as a regulator of the Na /Ca ‐exchanger activity in cardiomyocytes 2) Explore whether Na+/K+ ATPase coupling to ankyrin B regulates the activity of the Na+/Ca2+‐ exchanger in cardiomyocytes

2+ 3) Investigate whether inhibition of ICaL can reduce SR Ca content and prevent development of Ca2+ waves with special focus on the ankyrin B syndrome

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.

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.

3.3 Electrophysiological methods

3.3.1 Voltage clamp Whole cell voltage clamp is a commonly used technique for studying electrical activity of cardiomyocytes, and the technique was used in all three papers of this thesis. In this technique, the voltage (membrane potential) is clamped, while current from ion transporters or channels is measured. Briefly, a cell is impaled with an electrode and electrical activity is measured relative to a reference electrode in the cell bath. In more details, a glass electrode/pipette with a tip diameter of only a few micrometers and filled with an ionic solution similar to the intracellular environment is pressed gently onto the cell membrane to form a connection between the pipette and the cell membrane. This connection is called a gigaohm‐seal because of the high electric resistance. When suction is applied to the pipette, the negative pressure causes the underlying membrane patch to break. A ground electrode is placed in the extracellular fluid and hence, a closed electric circuit is established. In theory, this implies that any electrogenic current across the cell membrane will result in an equal current going through the pipette. The latter current can be registered and measured. By convention, any positive current is a positive current out of the pipette. This corresponds to a positive outward current from the cell (net extrusion of positive charges). ICaL (measured in paper 3) is an inward directed positive current, and will therefore be recorded as a negative current. NKA current (INKA) and NCX current (INCX) (measured in paper 1 and 2), on the other hand, takes net positive charge out of the cell (NKA: 2 K+ in, 3 Na+ out; NCX – reverse mode: 1 Ca+ in, 3 Na+ out), and are recorded as positive currents. To study specific ion transporter currents, there are in general three options: 1) pharmaceutical manipulation, 2) manipulation of membrane potential, and 3) regulation of substrate availability. We used combinations of these three approaches in our experiments. ICaL was activated by a 100 ms square pulse from ‐45 mV to 0 mV at 1 Hz stimulation rate. The extracellular solution contained 4‐ aminopyridine, Ba2+ and tetraethylammonium (TEA) to block K+ channels. After a voltage step, a capacitive current will follow to “recharge” the membrane. As ICaL‐peak is reached within 2‐7 ms, it is crucial to have a good voltage control of the cell. Poor voltage control would tend to underestimate the ICaL peak. To account for this, we used the discontinuous voltage clamp mode (switch clamp), where the amplifier switches rapidly between measuring membrane potential and injecting current (switch rate 9 kHz). We voltage clamped single rod shaped cardiomyocytes with apparently intact cell membrane. Whole cell voltage clamp is a technique to study cardiomyocytes in vitro. This means that, with isolated voltage clamped single cardiomyocytes, one can get a precise control of membrane potential and ionic composition in both intracellular and extracellular solutions.

3.3.2 Protocol for NKA dependent regulation of NCX

For the INKA and INCX measurements in paper 1 and 2, we used low resistance pipettes. This allows cell dialysis exchanging the cytosol with internal solution in the pipette. Because the volume of the pipette solution is much higher than the volume of the cytosol and a negative pressure is applied to the pipette, a high degree of cell dialysis is expected to happen within minutes after initiating the experiment.

+ 2+ INKA and INCX was activated by adding ions to the superfusate (K and Ca , respectively), with

+ + 2+ inhibitors of ICaL (nicardipine) and K channels (Cs and Ba ) present as illustrated in figure 5. The holding potential of –50 mV ensured inactivation of Na+ channels. In these experiments, we used continuous voltage clamp mode of a Axopatch 200B amplifier, as this is less noisy than the Axoclamp

2B amplifier with the possibility of discontinuous voltage clamp mode. This aspect is important as INKA and INCX are relatively small compared to i.e ICaL. Control of rapid voltage alterations was not an issue as INKA and INCX were activated by regulating substrate availability, and not by voltage alterations, which would require discontinuous voltage clamp. In subset of experiments in paper 2, we aimed to study the role of NKA‐binding to ankB by administration of specific peptide sequences containing an ankB‐binding site of NKA, as described more thoroughly in the method section of paper 2. A scrambled peptide sequence with the same length and randomized amino acid order was used as control in separate experiments. Results from experiments with the scrambled peptide were compared with results obtained in a comparable experimental setting without any peptides, and comparable results from experiments without peptide and the scrambled peptide were interpreted as no unspecific effects induced by the scrambled peptide. In all experiments where the peptide effect was examined, cells were compared with separate cells from the same experimental day treated with scrambled peptides.

Figure 5: Schematic protocol for studying NKA‐ and NCX‐activity. Taken from paper 1.

3.3.3 Methodological considerations regarding NKA dependent regulation of NCX The protocol for NKA‐dependent regulation of NCX‐activity is central for the conclusions in this thesis. An important aspect with the applied protocol is that the global cytosolic concentration of all ions were kept almost constant, as low resistance pipettes with a fixed internal solution were used, aiming for a constant dialysis of the cell in a setting with clamped membrane potential at ‐50 mV. Hence, both intracellular and extracellular Ca2+, the membrane potential and extracellular Na+

+ were kept constant during the experiment, meaning that the Na c sensed by the NCX would be the only unknown parameter affecting the NCX‐activity during the protocol. Based on these assumptions, we have in paper 1 and 2 of this thesis interpreted the reverse mode NCX as a sensor of Na+ at the cytosolic site of NCX. By application of K+ to the superfusate in this protocol, we and others typically observed a gradual decline in INKA over several minutes. This gradual decline is interpreted to be caused by gradual Na+ depletion due to NKA‐activity, although oxidative modifications of NKA might be a regulatory mechanism also influencing the observed INKA ‐decline during the protocol [120]. We

+ observed the decline in INKA in paper 2 to increase both in time and amplitude with increasing Na  in the experiments when the cells were superfused and dialyzed with the same Na+. Our explanation of this phenomenon is that the decline in INKA in this protocol is caused by gradual depletion of

+ + cytosolic Na as a result of active INKA until a steady state with balanced Na leak and extrusion is reached.

3.3.4 Field‐stimulation In order to trigger Ca2+ transients in paper 2 and 3, Ca2+ waves and Ca2+ sparks in paper 3, and cardiomyocyte contraction in paper 1, we field‐stimulated cardiomyocytes by passing current in the fluid surrounding the cell. This method has a higher success‐rate than voltage clamp experiments, which would be an alternative way to stimulate cardiomyocytes. Also, it is reasonable to argue that field stimulation elicit AP‐like events, as opposed to the less physiological voltage step stimulus in voltage clamp. Still, the APs triggered by field stimulation probably differ from the in vivo situation [121], where contractions are triggered normally by APs from the SA‐node spreading from cell to cell. In paper 1, we measured cardiomyocyte contraction as fractional shortening in field stimulated cardiomyocytes using a video edge detection system. The isolated cardiomyocytes were plated on laminin, a glycoprotein and a component of the basal lamina. Laminin promotes adhesion of the cardiomyocytes to the glass cover slips. This might interfere with the contractile properties. In vivo, cardiomyocyte contractions are affected by mechanical forces (preload and afterload), whereas with

31 this technique, the measured contractions are unloaded. Hence, these results cannot be directly extrapolated to the in vivo situation.

3.4 Immunocytochemistry

Immunocytochemistry was used to study the subcellular distribution of NKAα1 and ‐α2 in paper 1. Fixated and permeabilized cardiomyocytes were incubated with primary antibodies against the

NKAα1 and ‐α2. Fluorochrome‐conjugated secondary antibodies were then applied to bind to the primary antibodies and hence used to visualize the localization of the NKAs with a confocal microscope. To use confocal imaging to determine protein colocalization in restricted membrane areas is difficult due to limited optical resolution. Subsequently, we only used the results obtained with this method to determine NKA localization between the t‐tubules versus the surface sarcolemma. The specificity of the antibodies is crucial for obtaining high quality immunostains. Still, an important issue is unspecific binding of the primary antibodies. To avoid this, the cells were incubated in goat serum to reduce the number of epitopes available for unspecific binding. Furthermore, the fixation‐ and permeabilization procedures might influence the integrity and availability of the epitopes. Hence, it is important to optimize the labelling protocols, and this is in part based on experience and empirical trial and failure. Still, we were careful to set conditions equal in comparable experiment series to avoid variations in the images due to differences in labelling protocols.

3.5 Detubulation Immunocytochemistry can give information about protein distribution in the t‐tubules, but can not give information about t‐tubule function. In paper 1, we detubulated normal cardiomyocytes with formamide (HCONH2), using a technique described by Kawai et al [122]. The aim of this procedure was to isolate the surface membrane from the t‐tubule membrane. Formamide is membrane permeant. Hence, the substance will over time enter the cell down its concentration gradient. So when 1.5 M formamide is added to a solution with cardiomyocytes, this increases the osmotic pressure of the solution from 286 mosmol to 1780 mosmol. This osmotic change causes an initial decrease in cell volume since water will flux from the cells and to the solution (figure 6, 1). As formamide is membrane permeant, the substance will over time enter the cell down its concentration gradient. Water will follow, and the cell volume normalizes in the continued presence of formamide. Upon reapplication of the control solution/removal of formamide,

32 the intracellular concentration of formamide is initially high, meaning that water will enter the cell. This causes a rapid cell swelling (the osmotic shock) (figure 6, 2) that breaks the t‐tubules from the sarcolemma. Over time, formamide will leave the cell down its concentration gradient, water will follow, and cell volume and shape will normalize. Formamide is a denaturant and could affect protein‐protein interactions and distribution of membrane proteins. There is no direct proof that NKAs are not redistributed or otherwise altered following formamide exposure, as discussed in paper 1. Hence, we are cautious about potential formamide effects, and our conclusions in paper 1 based on results from detubulated cardiomyocytes are limited to distribution of NKAs in the surface membrane versus the t‐tubules.

1 2

Figure 6: Cell volume over time during formamide exposure. Figure taken from Kawai et al [122] with permission.

3.6 Peptide pulldown assay In paper 2, we used pulldown assays to determine whether the synthesized NKA MAB‐peptide is able to coprecipitate with endogenous ankB as an output of ankB affinity for the synthesized peptide. For these experiments, the MAB‐peptide was biotinylated and added to LV lysates, incubated and subsequently subjected to biotin immunoprecipitation as described in the method section of paper 2. The immunoprecipitate was immunostained for ankB, and higher levels of ankB in MAB pulldowns than scrambled pulldowns suggested that the synthesized MAB bound to endogenous ankB. Thence, we anticipated the same effect in the patch clamp experiments using the same peptide in the internal solution, ie. that the MAB‐peptide in these experiments binds to the ankB‐complex in the voltage clamped cardiomyocytes. In this thesis, we have not further studied the molecular effect of adding the MAB‐peptide to the ankB‐complex, which could potentially be due to more than one biological effect, including disruption of the NKA‐ankB interaction or MAB‐binding to ankB not coupled to NKA.

3.7 Fluorescence microscopy Fluorochromes are substances that, upon light exposure of a certain wavelength, reemit light of a longer wavelength. Different fluorochromes can bind to different structures or substances, and some fluorochromes have a specific affinity for ions like Na+ and Ca2+. In a fluorescence microscope, the emitted fluorescence is separated from the illumination light by the use of an emission filter. We used fluorescence microscopy in all three papers of this thesis, but for different purposes:

a. To visualize cell membrane labeling b. To record Ca2+ transients

+ c. To measure Na c a. In paper 1 and 2, the cell membrane was labeled with the fluorochrome di‐8‐ANEPPS. Compared to other ANEPPS‐dyes, an advantage of using di‐8‐ANEPPS is that this substance is more photostable, less phototoxic and less susceptible to internalization [123], reducing unwanted labelling of intracellular organelles. b. In paper 1 and 3, we recorded Ca2+ transients in field stimulated cardiomyocytes stained with fluo‐4 acetoxymethyl (AM) esther. The AM‐esther can cross cell membranes. Hence, the cells can be loaded with dye by applying it to the extracellular solution. In cytosol, a deestherification with removal of lipophilic groups takes place, so that the compound is less likely to leak back through the cell membrane or into intracellular organelles, such as the mitochondria. We loaded cells at room temperature, which is thought to slow the loading speed and potentially leaving time for deestherification. In paper 3, we also recorded Ca2+ transients in voltage clamped cardiomyocytes using fluo‐5F (salt) applied in the pipette/intracellular solution. c. In paper 1, sodium‐binding benzofurzan isophtalate (SBFI) (AM‐esther) was used to measure

+ resting global Na c in quiescent rat cardiomyocytes. Many laboratories use SBFI to measure of Na+ in the dual excitation ratio mode(340 nm/380 nm) and single emission (500 nm). We use a method described first by Baartscheer [124], with single excitation (340 nm) and dual ratiometric emission (410nm/590nm). An advantage with this method is a greater ability to detect small

+ changes in Na c. However, a disadvantage is a less precise measure of high concentrations. Use of SBFI does not permit detection of sub‐sarcolemmal Na+ domains in the nanometer range, far below the detection limit for confocal‐ and fluorescence microscopy. Furthermore, Na+ binds

+ slowly to SBFI. Hence, rapid and transient changes in Na c are not likely to be reflected in the SBFI‐signal.

3.8 Ca2+ imaging with confocal microscopy In paper 3, Ca2+ sparks and Ca2+ waves were evaluated using linescan imaging by confocal microscopy. Confocal imaging provides a detailed in depth look into the cell, but acquiring a full scan of the cell takes a relatively long time and repetitive pictures may lead to photobleaching of the cell. Linescan imaging, where fluorescence along a line through the cell is recorded, is a better way to evaluate quick events like the spread of cytosolic Ca2+. It also reduces photobleaching as a smaller area of the cell is illuminated.

3.9 Western blot Western blot analysis can detect one type of protein in a mixture of any number of proteins and give information about the size and relative amount of the protein. Gel electrophoresis is used to separate the different proteins by their respective mass. The proteins are then transferred out of the gel and onto a polyvinylidine diflouride (PDVF) membrane. This membrane is incubated with a primary antibody against the proteins of interest. A secondary antibody coupled to a conjugated enzyme is applied to bind to the primary antibody. By use of enhanced chemiluminescence, the complex of the protein, primary antibody and secondary antibody can be visualized. In paper 1 and 2, Western blot was used to measure protein abundance. In paper 3, hearts were perfused with isoproterenol +/‐ verapamil and Western blot was then used to determine the effect of verapamil on phosphorylation of Ca2+ handling proteins. In general, western blot is a semi‐quantitative method and cannot be used to calculate protein concentrations. Hence, small changes (less than 15‐20%) in labelling intensity should be carefully interpreted.

3.10 Computer models The main strength of cell experiments with patch clamp techniques is a direct measurement of the activity of one specific ion channel or transporter. The interplay between two or different channels is more difficult to investigate directly. Mathematical models are often used as a supplementary tool to simulate multiple ion fluxes based on experimental data as input. In paper 3, computational

2+ modelling was used to study the effect of ICaL on SR Ca content, and more specifically to predict

2+ whether the verapamil mediated reduction in ICaL could be a sufficient factor to reduce SR Ca content. In this study, we applied a previously published mathematical model of intact cardiomyocytes, based on parameterization of several ion channels, including LTCC, RyR, SERCA2, NCX, K+‐channels and Na+‐channels. One strength of this model is the extensive validation of various

35 cell electrical phenotypes in experimental settings, as the model has been used in several other studies comparing experimental and mathematical data [125‐129]. In future, computational modelling might also increase the understanding of another main question in this thesis, namely the existence and importance of cytosolic Na+ gradients. To our knowledge, there is at present no mathematical model of cardiomyocytes taking Na+ gradients inside the cell into account.

4. Summary of results The experiments in paper 1 were performed in rat cardiomyocytes, in paper 2 in rat and mouse cardiomyocytes, and in paper 3 in mouse cardiomyocytes.

4.1 Paper 1 In paper 1, we used a combination of immunocytochemistry, cellular electrophysiological techniques and fluorescence microscopy to study the functional roles of the two main NKA isoforms in rat hearts, NKA 1 and 2. We first found that the 1‐isoform was significantly less abundant in the t‐ tubules than in the surface membrane, whereas the 2‐isoform was more abundant in the t‐tubules than in the surface membrane. A dose‐response curve for oubain was obtained to determine the relative oubain sensitivity of NKA1 versus‐ 2. A concentration of 0.3 µM ouabain blocked ~94% of the 2‐isoform, but less than 1% of the 1‐isoform. Hence, this dose of ouabain was used to study 2‐ isoform function in the later experiments. By application of 0.3 µM of ouabain, we detected a 10.7 

0.6% reduction of INKA, indicating that ~10% of the NKAs in rat cardiomyocytes are constituted by

NKA2. In detubulated cells, 0.3 µM ouabain had a smaller effect (6.0  0.5%) on INKA, indicative of a higher density of NKA2 in t‐tubules than in the surface sarcolemma. Furthermore, 53% of the INKA,2, but only 9.5% of INKA,1 was located in the t‐tubular membrane, indicating that the INKA,2 was functionally located to the t‐tubules.

Next, the interaction of NKA2 with NCX was assessed functionally. Exposure to 0.3 µM

+ + ouabain increased subcellular [Na ]c which was assessed by INCX, indicating an increase of [Na ]c in the

+ submembrane compartment of about 3‐5 mM. However, no increase in global [Na ]c was detected. In line with these observations, 0.3 uM ouabain had a significant inotropic effect in field stimulated cardiomyocytes, and increased myocyte shortening by 40% and Ca2+ transients by 70%,

2+ demonstrating NKA2 as a regulator of contractility and Ca transients. Overall, the results suggest that NKA2 controls NCX activity within a restricted microdomain within the t‐tubules.

4.2 Paper 2 In paper 2, we used a combination of the MAB‐peptide and ankB+/‐ deficient cardiomyocytes to study NKA coupling to ankB as a potential regulator of NCX‐activity in cardiomyocytes. We first verified the ability of the MAB‐peptide to couple to ankB by pulldown experiments with LV lysates. To be able to study whether the MAB peptide alters NKA transport kinetics, we firstly established a protocol with the same Na+ in the internal solution and superfusate to avoid the bias of transmembrane Na+

+ gradients. By eliciting INKA repetitively with addition and removal of K in the superfusate, we established that the peak INKA after the second and subsequent elicitations of INKA most likely reflects

+ + the Na dependent activity of NKA. The MAB peptide did not alter INKA at [Na ]c = 17 mM, but

+ significantly increased INKA at high [Na ]c (80 mM). These results provided an important basis for our next experiments designed to evaluate the role of ankB coupling of NKA as determinant of NCX

+ function. In a protocol designed to study NKA dependent regulation of [Na ]c sensed by the NCX, the

MAB peptide increased the INCX and also abolished the correlation between INKA and INCX observed in control experiments. Based on these observations, we suggest a model where 1) ankB coupling of

+ + NKA lowers the [Na ]c sensed by the NCX and 2) that NKA and NCX sense the same subcellular Na c due to ankB‐coupling. This model was further supported by the observation that INKA and INCX correlated in WT cardiomyocytes, but not in ankB‐deficient cardiomyocytes. Similar to these findings in quiescent cells, we lastly observed that TAT‐conjugated (cell permeable) MAB‐peptides reduced NCX mediated Ca2+ extrusion in beating cardiomyocytes, consistent with the MAB peptide to increase

+ Na c sensed by the NCX. Altogether, these results suggest that ankB orchestrates a macromolecular complex where NKA regulates NCX activity within a shared subcellular microdomain.

4.3 Paper 3 In paper 3, we investigated a role of the CCB verapamil as a potential antiarrhythmic treatment. Ca2+ waves were induced in cardiomyocytes by isoproterenol combined with caffeine to increase RyR Ca2+

2+ 2+ sensitivity. ICaL inhibition by verapamil reduced the probability for Ca waves, frequency of Ca sparks and SR Ca2+ content in cardiomyocytes pretreated with isoproterenol and caffeine. In permeabilized cardiomyocytes, verapamil had no effect on Ca2+ sparks confirming that the observed effects on Ca2+ handling were not a result of direct effects of verapamil on RyR. Furthermore, in separate experiments with perfusion of excised hearts, verapamil did not change the phosphorylation status of neither phospholamban (Ser16, Thr17) nor RyR (Ser2809, Ser2814). The experimental data were supported with mathematical modelling, that concluded that inhibition of LTCC was sufficient to reduce SR Ca2+ load. In ankB+/‐ cardiomyocytes, we found that verapamil reduced the frequency and probability for Ca2+ waves, suggesting that Ca2+ channel blockade may be a beneficial clinical treatment strategy to prevent arrhythmias in patients suffering from the ankyrin B syndrome.

5. Discussion

5.1 Subcellular distribution of NKA1 and ‐2 isoforms

In paper 1, using immunocytochemistry, we found that NKA1 was significantly less abundant in the t‐tubules than in the surface membrane, whereas the NKA2 was more abundant in the t‐tubules than in the surface membrane. An earlier study performed in cardiomyocytes from guinea‐pig hearts showed the same distribution of 1‐and 2‐isoforms in the surface sarcolemma and the t‐tubules [130]. Other studies using immunocytochemistry have shown different results. In rats, studies have reported 1) uniform distribution of 1‐isoform and 2‐isoforms in the surface sarcolemma, with low staining of both isoform in t‐tubules [47], and 2) uniformly distributed 2‐isoforms in surface sarcolemma and t‐tubules, and 1‐isoforms mostly in t‐tubules [131]. Use of different antibodies with different specificity and different accessibility for epitopes in the surface sarcolemma and t‐tubules might explain these apparently diverging results.

To examine whether NKA2 was also functionally preponderant in the t‐tubules, we measured INKA in control and detubulated cardiomyocytes. In these experiments, we made use of the difference in affinity for ouabain between the 1‐ and 2 isoform observed in rodents. A dose‐ response curve for ouabain was established. This gave us the dose of ouabain required to specifically block NKAα2. 0.3 µM ouabain blocked ~94% of the 2‐isoform, but less than 1% of the 1‐isoform, and this low dose of ouabain was used to study 2‐isoform function. Other investigators have used a similar approach and found comparable results [132]. When control cardiomyocytes were exposed to

0.3µM of ouabain, INKA density was reduced by 10.7  0.6%. The reduction in INKA density induced by the low concentration of ouabain was smaller after detubulation (6.0  0.5%), indicating a higher density of 2‐isoforms in the t‐tubules versus the surface sarcolemma. These data are in line with findings in mice (12% and 6%, respectively) [133] and in the range with other findings in rat cardiomyocytes (30% and 18%, respectively) [134]. Both studies [133] and [134] conclude that the functional density of NKA2 isoforms is 4‐5 times higher in the t‐tubules versus the surface sarcolemma.

Accordingly, we conclude that the INKA,2 is predominately located in the t‐tubules, both based on functional and imaging data.

5.2 NKAα2 controls NCX‐activity + In paper 1, we hypothesized that NKA2 might regulate Na c and thus also NCX‐activity in a specific subcellular compartment near the t‐tubules. We applied a whole‐cell voltage clamp protocol to study

NKA‐dependent NCX‐activity. The main conclusion from these experiments is that NKA2 is a

+ regulator of NCX‐function by affecting the Na  sensed by NCX on the cytosolic site. These findings are in line with the prior investigations of NKA2 deficient cardiomyocytes, which showed larger INCX

+ when Na was rapidly removed from the superfusate than wild type and NKA1‐deficient cardiomyocytes [135].

We also found (in paper 1) that NKAα2 only constituted ~10% of the total INKA in rat cardiomyocytes. Still, specific inhibition of NKAα2 resulted in a profound positive inotropic effect

+ without an observed increase in global Na c. We detected a potent ability of NKAα2‐isoforms to control NCX‐activity, and hypothesized that NKAα2 controls NCX activity through a common

+ subcellular domain or pool. A key line of evidence supporting this idea is the unaltered global Na c

+ detected using the Na sensor SBFI. These data support a model where NKA2 does not have a

+ + significant role in regulating the global Na c, but controls local pools of Na in the vicinity of NCX.

+ However, the unaltered global Na c observed during NKAα2 inhibition in our study is in some contrast to what was reported by Despa et al [132], where a comparable approach in mice

+ cardiomyocytes treated with a comparable dose of ouabain showed an increase in Na c of ~2 mM,

+ measured with SBFI. We calculated that the detection limit of SBFI for changes in Na c was 1.7 mM

+ in paper 1, implying that Na c in cardiomyocytes could be increased with up to 1.7 mM in our

+ experiments without being detected by our assay. As even a minor elevation of Na c can induce significant inotropic effects in cardiomyocytes [31, 32], it is possible that NKA2 is not functionally

+ coupled to NCX, as reported in paper 1, but rather controls the global Na c and thus INCX only indirectly. We calculated that NKA2‐inhibition by the low dose of ouabain would cause an increase in local Na+ of 4‐5 mM, well above our detection limit, but presumably not enough to increase the

+ Na c to a level detectable by SBFI. Finally, a methodological consideration for the interpretation of

+ these experiments is that we have used SBFI to measure Na c in the single excitation/dual emission

+ + configuration [124], reported to give a more precise measurement of Na  close to values of Na c than the approach with dual excitation/single emission more often used, i.e in [132].

A main conclusion in paper 1 is thus that NKA2 seems to regulate a subcellular domain of Na+ in the vicinity of NCX. This conclusion is also supported by a later study from our group, where the reduction in INKA closely correlated with the reduction in INCX in sham operated rats, while this correlation was abolished in rats with post‐myocardial infarction heart failure with a major

+ downregulation of NKAα2 [136]. Although a common pool of Na shared between NKA2 and NCX

40 remains to be directly demonstrated, we conclude that a functional interaction seems likely to exist between the two transporters.

5.3 The MAB‐peptide ‐ a disruptor peptide of the NKA‐coupling to ankB In paper 2, we studied the potential role of ankB coupling of NKA as an underlying molecular mechanism for creating a shared microdomain between NKA and NCX. In this study, we first synthesized the MAB peptide previously demonstrated to constitute the main ankyrin interaction site on the α subunit of NKA. This sequence has earlier been reported to have minimal or no overlap with any of the other known ankyrin binding proteins [79], providing a potential specific disruptor peptide of the NKA‐ankyrin coupling. However, there are at least two unknown factors regarding the ability of the MAB peptide to disrupt NKA from ankB:  Specificity for ankB: While ankB seems to be a key anchoring molecule for NKA and NCX in cardiomyocytes, the MAB sequence on NKA has only directly been demonstrated to show affinity for ankyrin G and ankyrin R, both present in cardiomyocytes. To our knowledge the ability of the MAB sequence on NKA to couple to ankB has not been tested previously, and our demonstration that biotin tagged MAB couples to endogenous ankB in pulldown experiments suggests that the MAB sequence may be the ankB coupling site on the NKA‐isoform. An unknown factor in these experiments is whether NKA in cardiomyocytes also binds to ankyrin G and ankyrin R (as in other cell types) in addition to ankB, thus disrupting NKA from all ankyrins with unknown relative affinity for each interaction.  Disruptor properties of the MAB peptide: Although we have demonstrated that the MAB peptide coprecipitates with endogenous ankB in pulldown experiments, suggesting that the peptide has an affinity for the ankB‐complex, the ability of the peptide to disrupt NKA from ankB is unknown. Firstly, NKA is shown to interact with ankyrin G and ankyrin R both with the 2nd and 3rd intracellular loop (CD2 and CD3) [75], meaning that even complete disruption of the ankB coupling to the MAB‐sequence does not completely uncouple NKA from ankyrin. Secondly, the concentration‐disruption relationship between the MAB‐peptide and NKA‐ankB is not known. As we detected physiological effects with 1 µM peptide in the internal solution or the superfusate, we expect this peptide concentration to at least partly be able to disrupt NKAs coupled to ankyrins in the cell.

Based on earlier reports on the MAB sequence and our demonstration of biotin tagged MAB to couple to endogenous ankB in pulldown experiments from LV‐lysates, we have interpreted the functional data obtained with this peptide as NKA‐disruption from ankB.

5.4 AnkB as basis for NKA dependent regulation of NCX To investigate how coupling of ankB to NKA functionally regulates NCX activity in cardiomyocytes, we firstly aimed to determine whether the Na+ dependency of NKA was altered by the MAB‐peptide.

+ + Patch clamp experiments with variable Na  in the pipette are often used to obtain Na dependency curves of the NKA. This approach could, however, underestimate the submembrane Na+, as this

+ could be higher than the Na  in the pipette [87]. In paper 2, we thus designed a protocol with

+ + similar Na  in the superfusate and the internal solution to avoid transmembrane Na leak, which probably could give a more direct measurement of the Na+ dependency of NKA. By use of this

+ protocol, we surprisingly found that the MAB‐peptide increased INKA at Na c = 80 mM. These data indicate that NKA‐coupling to ankB inhibits the maximal NKA‐transport capacity. We next examined whether the MAB peptide altered the NKA dependent control of NCX activity, using the protocol schematically shown in Figure 5. In this protocol, both INKA and INCX can be

+ viewed as output of the Na  sensed by NKA and NCX given that none of the other parameters, such

+ 2+ as the extracellular K or Ca concentrations, are altered. We did not detect any differences in INCX before activation of INKA and increased INCX after activation of INKA during MAB‐peptide exposure. Hence, these results support the idea that ankB provides a functional coupling of NKA to NCX.

We detected increased INKA within the same protocol, which in theory given specificity of the

+ + MAB‐peptide, could be due to either increased Na c at the cytosolic site of NKA, increased Na sensitivity of NKA or increased Na+ leak induced by the MAB‐peptide. As we also found that the NKA

+ activity at high [Na ] was higher in cells treated with the MAB‐peptide, the INKA in this setting could not be used as an output of the Na+ sensed by the NKA. We thus cannot conclude based on this

+ result alone that exposure to the MAB‐peptide increases INCX through modulation of a subcellular Na

+ domain since this effect could also be caused by increased global Na c.

Therefore, the demonstration of a correlation between reductions in INKA and INCX observed in control experiments, but not in MAB‐treated cells, is important for the conclusions of this study. The demonstration of a correlation between reductions in INKA and INCX are consistent with the two proteins sensing the same Na+ pool in the cardiomyocytes in the control experiments. If the MAB‐

+ peptide exerted its effect by simply increasing the Na  in the cell, and there was no subcellular interaction between the two transporters, a correlation between INKA and INCX would be expected in the MAB treated cell. This was not the case, and we did not detect a correlation between INKA and INCX in MAB treated cells, suggestive of a shared NKA and NCX microdomain orchestrated by ankB. To support the results obtained with the MAB‐peptide, we also applied the same protocol in ankB+/‐ cardiomyocytes. AnkB+/‐ mice have, as discussed in the introduction, lower levels and altered subcellular distribution of NKA [78]. Hence, ankB+/‐ cardiomyocytes were primarily used to examine

42 whether the correlation between INKA and INCX was abolished also in these cells. Further supportive of our working model, we detected a significant correlation between INKA‐ and INCX‐reductions by application of the same whole cell voltage clamp protocol in wild type, but not in ankB+/‐ cardiomyocytes. We conclude that these experiments collectively support a model where ankB

+ coupling of NKA 1) regulates the Na  sensed by NCX and 2) allows regulation of NCX activity within a shared subcellular domain.

5.5 NKA‐regulation of Ca2+ fluxes through control of NCX‐activity In summary, results from paper 1 and 2 in this thesis is in line with the existence of two NKA‐

+ mediated determinants of the Na c sensed by the NCX, namely by functional coupling to NKA2 and anchoring to ankB. As the NKA2‐isoform was clustered in the t‐tubules, we hypothesized that the

NKA2‐isoform controls the ECR‐coupling and contractility by modulation of NCX‐activity. This was supported by the findings in paper 1, where we exposed contracting cardiomyocytes to the NKAα2‐ selective dose of ouabain and found a significant inotropic effect (40% increased FS/TTP) and

2+ increased Ca transient amplitude, demonstrating that NKAα2 exerts modulation of NCX‐activity and ECR‐coupling in regular cardiomyocytes. These observations are in line with the hypercontractile function of NKA2 KO‐mice [54] and a comparable study where a low dose of ouabain was applied both in regular mice and mice with genetically swapped low ouabain sensitivity from the NKAα2‐ isoform to the α1‐isoform (SWAP mice) [132]. An important finding in the latter study was that if the same percentage of NKA1‐pumps (using SWAP‐mice) was inhibited by a low dose of ouabain as

+ inhibited in controls, Na c was increased to the same level as with low dose ouabain administration to control mice. Thus, based on this study, NKAα1 and ‐α2 exert a comparable degree of control of the

+ 2+ global Na c. An important next question in this study was whether NKA2 regulated Ca fluxes more tightly than NKAα1. In line with our results, a key finding was the demonstration of a significant

2+ increase in Ca transients only by inhibition of NKA2‐isoforms, but not by inhibiting the same percentage of NKA1 in SWAP mice. These observations clearly support that NKA2 regulates NCX more closely than NKA1.

The molecular determinant of the NKA2‐mediated interaction with NCX is not known. This could in theory involve colocalization within the same macromolecular complex, as NCX coimmunoprecipitates with NKA2 [137]. However, this is not a straight forward explanation, as also

NKA1 coimmunoprecipitates with NCX [138]. As ankB has been shown to be necessary for the immunoprecipitation of NCX to both isoforms of NKA [92], it is tempting to speculate that NKA2 has a higher affinity to the NKA anchoring site at ankB. If so, NKA2 will be more abundant in the vicinity of NCX than NKA1. Detailed studies of the protein affinities between ankB and NKA would be of

43 great interest to get closer to the molecular mechanisms allowing the NKA2‐dependent control of NCX.

Another unsolved question is the subcellular distribution of the proposed NKA2‐NCX complex. NKA2 is abundant in the t‐tubules, but the position of NKA2 relative to the dyad in cardiomyocytes is not currently known. In smooth muscle cells and astrocytes [139], a close interaction between NKA2 and ER has been demonstrated, highlighting a possible localization of

2+ NKA2 close to the dyadic cleft and thus a role for NKA2‐NCX interaction in controlling Ca fluxes

+ during ECR‐coupling. By such localization, inhibition of NKA2 would tend to increase Na  close to

2+ the dyad, meaning that the reverse mode INCX would be increased and perhaps trigger more Ca release during a twitch. Another possibility would be a preferentially extradyadic localization of

NKA2‐NCX, where 2‐inhibition would tend to decrease NCX forward mode activity and thus lead to increased SR Ca2+ load by shifting the SERCA2‐NCX balance during Ca2+ extrusion in favor of SERCA2. Such a role has been supported by a recent publication by our group, where flash photolysis of caged

+ 2+ 2+ Na to measure Ca extrusion supported a role for NKA2 in controlling NCX‐mediated Ca extrusion in cardiomyocytes [140]. In line with the idea that NKA regulates ECR‐coupling by specific subcellular localization, we observed in paper 2 that TAT‐conjugated (cell permeable) MAB‐peptides reduced NCX mediated Ca2+ extrusion. This firstly supports the model proposed in the previous section on ankB coupling of NKA as a regulator of Na+ levels sensed by the NCX in another assay, and secondly demonstrates that this effect is present also in beating cardiomyocytes to highlight a physiological relevance of this interaction. These data are in line with data from Camors et al, reporting lower NCX mediated Ca2+ extrusion in ankB deficient cardiomyocytes [111]. As the study by Camors et al concluded that ankB mediated NCX activity regulated RyR conductance by localization in the dyad, we speculate that the ankB coupling of NKA regulates reverse mode NCX activity and RyR conductance in addition to our demonstration ankB dependent regulation of forward mode NCX activity, but further experiments will be necessary to confirm this idea.

In conclusion, we propose a model where NKA2 regulates NCX‐activity in cardiomyocytes by controlling a localized pool of Na+, as investigated in paper 1. As investigated in paper 2, NKA‐ coupling to ankB provides a control mechanism for NCX‐activity in a comparable manner. Future studies might show whether ankB preferentially binds NKA 2 and the relative (co)localization of ankB, NKA2 and the dyad.

5.6 Verapamil prevents Ca2+ waves in ankB+/‐ cardiomyocytes One aim in this thesis was to examine whether CCBs are able to prevent Ca2+ waves, especially in ankB+/‐ cardiomyocytes. In paper 3, the commonly used CCB verapamil was tested in both caffeine‐ treated cardiomyocytes (to evoke Ca2+ waves as a result of increased RyR opening probability) and in ankB+/‐ cardiomyocytes. The main result in this study was that verapamil was able to significantly

2+ reduce the amount of cells with Ca waves in both models. Exposure to verapamil reduced the ICaL and SR Ca2+content, but did not alter RyR‐phosphorylation status. Thus, it is reasonable to link the ability of verapamil to prevent Ca2+ waves to its ability to reduce SR Ca2+ load, rather than altered RyR‐opening probability. Our interpretation of the data in paper 3 is that intrinsic RyR properties are not altered by verapamil. This is shown in figure 4 in the paper. A criticism of this interpretation could be that although the phosphorylation status of the RyR is not altered by verapamil in Langendorff perfused hearts, the applied cell permeabilization by saponin could disrupt the potential microdomains of Ca2+ close to the dyad. If the Ca2+ level in the dyad is regulated by NCX, in line with studies in NCX KO mice where NCX mediated Ca2+ influx was concluded to prime the dyadic cleft with Ca2+ prior to the opening of the LTCCs [19], the finding of unaltered RyR Ca2+ release by permeabilization of the cardiomyocyte might be biased. Permeabilization of cardiomyocytes in presence of verapamil could affect the local Ca2+concentration surrounding the RyR in the dyadic cleft, which in intact cells could be regulated by LTCCs and NCX. This role of NCX in priming the dyadic cleft with Ca2+ has been earlier investigated in ankB+/‐ cardiomyocytes [111]. Interestingly, in this study ankB+/‐ cardiomyocytes exerted increased Ca2+ spark tendency, due to an assumed increased Ca2+concentration in the dyad of ankB+/‐ cardiomyocytes. This conclusion was made based on the observation that permeabilization of the cell membrane abolished the increased Ca2+ spark tendency in ankB+/‐ cardiomyocytes. Importantly, in this study the SR Ca2+ load was controlled for, providing strong evidence that the increased tendency for spontaneous SR Ca2+ release in ankB+/‐ cardiomyocytes to be due to increased RyR‐conductance. Thus, an unexplored possibility in paper 3 might be that verapamil reduced Ca2+ waves by altering the Ca2+ concentration at the cytosolic site of RyRs, in addition to or rather than decreasing the SR Ca2+ load per se. This potential effect of verapamil in study 3 is not of merely theoretical interest, as verapamil in fact earlier has been shown to directly couple to purified RyRs [141], questioning the interpretation of the antiarrhythmic effect of verapamil to be due to SR Ca2+ load reduction in ankB+/‐ cardiomyocytes, as stated in paper 3. One bias might be that the frequency and characteristics of Ca2+ sparks in ankB+/‐ cardiomyocytes treated with verapamil was not examined in paper 3, as in an earlier study concluding that increased propensity for Ca2+ waves in ankB+/‐ is caused by altered RyR‐regulation [111].

Nevertheless, verapamil showed a significant ability to reduce Ca2+ waves in both caffeine treated cardiomyocytes and in ankB+/‐ cardiomyocytes, and one main conclusion in this thesis is thus that verapamil might have potential as antiarrhythmic therapy in patients with ankyrin B syndrome and also in other DAD‐induced arrhythmias, such as CPVT and digitalis intoxication. Yet, the ankyrin B syndrome has a complex phenotype, including induction of DADs, sinus node dysfunction, atrial fibrillation and CPVT‐like arrhythmias. Verapamil could affect other arrhythmogenic factors, such as reentry circuits, in an unknown manner not studied in paper 3 in patients with ankyrin B syndrome. A key to further understanding of the potential therapeutic role of verapamil in ankyrin B syndrome would be to investigate in vivo electrophysiological function in ankB+/‐ deficient mice treated with verapamil, as well as proper later clinical testing. In conclusion, verapamil might represent a new therapeutic approach to prevent arrhythmias in patients with ankyrin B syndrome by preventing DADs and triggering of tachyarrhythmias, such as ventricular tachycardia and fibrillation, but more studies are needed to gain further support of this idea in a clinical setting.

6. Conclusions

+ + + 2+ 1) The α2‐isoform of the Na /K ‐ATPase is functionally coupled to the Na /Ca ‐exchanger and can

2+ + regulate Ca handling without changing global [Na ]c. 2) Anchoring of the Na+/K+‐ATPase to ankyrin B provides the proximity to the Na+/Ca2+‐exchanger that allow for the functional coupling. 3) Calcium channel blockers represent a potential antiarrhythmic therapy in patients suffering from the ankyrin B syndrome.

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8. Errata

Introduction:

 List of abbreviations: IP3R should be written as “Inositol 1,4,5‐trisphosphate receptor”  Chapter 1.2.3, first paragraph, line 5‐6: The sentence should read “SERCA2‐activity is regulated by the short protein phospholamban (PLB), which in its unphosphorylated form inhibits SERCA2‐activity.”

Paper 2:

 Reference 10 and 14 are identical  Reference 17 refers to wrong journal and time of publishing, and should read: “Dostanic I,

Paul RJ, Lorenz JN, Theriault S, Van Huysse JW, Lingrel JB. The alpha2‐isoform of Na‐K‐ATPase mediates ouabain‐induced hypertension in mice and increased vascular contractility in vitro. J. Biol. Chem. 2003, 278:53026‐53034.”

9. Appendix: Paper 1‐3