The Sodium-Calcium Exchanger 1:NovelRegulatory Mechanisms and Interacting Partners

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NIVERSITY

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ACULTY OF

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The Sodium-Calcium Exchanger 1:

Novel Regulatory Mechanisms and Interacting Partners

T

HESIS SUBMITTED FOR THE DEGREE OF

P

HILOSOPHIAE

D

OCTOR

February 2017

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© Tandekile Lubelwana Hafver, 2017

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

ISBN 978-82-8377-025-4

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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Acknowledgements

The work for my PhD thesis was carried out from 2012 to 2016 at the Institute for Experimental Medical Research (IEMR), Oslo University Hospital, Ullevål and the University of Oslo, under the supervision of Dr. Cathrine Rein Carlson and Professor Ole Mathias Sejersted. Primary financial support was provided by Helse Sør Øst.

I would like to express my deepest gratitude to my supervisors, Cathrine Carlson and Ole Sejersted, for mentoring me throughout my PhD studies. Your knowledge, guidance and support have been invaluable to me. Throughout my time at IEMR I participated in national and international conferences and appreciate these valuable experiences.

I also wish to express my deepest gratitude to all the co-authors of the papers contained in this thesis. I am grateful for your contributions which have led to the completion of this work. I wish to thank all my colleagues at IEMR for their friendship, my office mates for a nice office environment, Lisbeth Winer and Jo-Ann Fabe Larsen for excellent administration, and the technical team for their support.

Lastly, I wish to thank my family and friends: To the Hafvers, thank you for all your support and encouragement throughout these years. To the Lubelwanas and my extended family in South Africa, thank you for your support, encouragement, enthusiasm and love. To my mother (Nontsikelelo Elizabeth Lubelwana née Sokopo Hermanus), your sacrifices over the years have not gone unnoticed.

Thank you for your support and love. To my husband Andreas, I would not choose anyone else to be with me on this journey. Thank you for your encouragement, the scientific discussions and joy you bring to my life. I am truly blessed to have you as my life partner.

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I dedicate this thesis to the memory of my grandparents, Siphokazi Mary Hermanus née Nkowane (23.10.1922 to 19.08.2005) and Rev. Dr. Jonathan Tim Hermanus (04.04.1919 to 12.05.2006), who have had an enormous influence in my life. My grandfather was an astute community leader (under the former Ciskei Government) who always put the interests of others before his. He was detained as a political prisoner in the infamous Robben Island prison (02.07.1969 to 01.07.1976) for his role in the liberation of his people in Apartheid South Africa. Despite his incarceration, this never discouraged him, and during this time he obtained his Bachelor of Theology (Th.B.) in 1971, Master of Theology (Th.M.) in 1973 and Doctor of Theology (Th.D.) in 1976, with the thesis entitled: “The arrival of missionaries in Africa, with special emphasis on the southern tip of Africa in the 19^thcentury”. He also went on to obtain a Doctor of Philosophy in Theological (PhD) in 1997. Jola, JoliNkomo, Qengeba, you serve as a source of inspiration, achieving against all odds.

‘uYehova ngumalusi wam’

Oslo, Norway, 6^thFebruary 2016 Tandekile Lubelwana Hafver

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Abbreviations

AB Aortic banding

ACE Angiotensin-converting enzyme

AM Acetoxymethyl

AS Aortic stenosis

Asn Asparagine

Asp Aspartic acid

ATP Adenosine triphosphate

BIAcore Biomolecular interaction analysis bpm Beats per minute

CABG Coronary artery bypass CaCA Ca²⁺/cation

CAD Coronary artery disease

CaMKII Ca²⁺/calmodulin dependent kinase II

CaMPDB Calpain for modulatory proteolysis database CBD Ca²⁺-binding domain

cDNA Complementary deoxyribonucleic acid CICR Ca²⁺induced Ca²⁺release

CLD Catenin-like domain Co-IP Co-immunoprecipitation

Cys Cysteine

DARPP-32 dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32,000

DAVID Database for annotation, visualization and integrated discovery DNA Deoxyribonucleic acid

FRET Fluorescence resonance energy transfer Glu Glutamic acid

GO Gene ontology

GST Glutathione S-transferase HEK293 Human embryonic kidney 293

HEPES N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic acid

HF Heart failure

His Histidine

His-TF- NCX1cyt

His-trigger factor-NCX1 cytosolic loop HPRD Human protein reference database

I-1 Inhibitor-1

I-2 Inhibitor-2

IP Immunoprecipitation

ITC Isothermal titration calorimetry

kDa Kilodalton

KEGG Kyoto encyclopedia of genes and genomes LTCC L-type Ca²⁺channel

LV Left ventricle

Lys Lysine

Met Methionine

mM Millimolar

mRNA Messenger RNA

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MS Mass spectrometry

NCX Sodium calcium exchanger

NKA Sodium/potassium-transporting ATPase

nM Nanomolar

NMR Nuclear magnetic resonance

Opt-pep NCX1-PLM disruptor peptide (developed in paper 3) PDB ID Protein data bank identification

Phe Phenylalanine

PI Phosphatidylinositol

PIP2 Phosphatidylinositol-4,5-bisphosphate PKA Protein kinase A

PKC Protein kinase C

PLA Proximity ligation assay

PLM Phospholemman

PLN Phospholamban

PMA Phorbol 12- myristate 13-acetate PMCA Plasma membrane Ca²⁺ATPase PNUTS PP1 nuclear targeting subunit PP1 Protein phosphatase 1

PP1c Protein phosphatase 1 catalytic subunit PP2A Protein phosphatase 2A

Pro Proline

pSer-68-PLM Phospho serine-68-phospholemman PVDF Polyvinylidene difluoride

RNA Ribonucleic acid RYR2 Ryanodine receptors 2

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Ser Serine

SERCA2 Sarcoendoplasmic reticulum Ca²⁺ATPase 2 SLC Solute carrier

SPR Surface plasmon resonance SR Sarcoplasmic reticulum

STRING Search tool for the retrieval of interacting genes TEV Tobacco etch virus

Thr Threonine

TM Transmembrane

Tyr Tyrosine

μM Micromolar

WT Wild type

XIP Exchanger inhibitory peptide YFP Yellow fluorescent protein

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List of papers

1. Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Pimthanya Wanichawan, Tandekile Lubelwana Hafver, Kjetil Hodne, Jan Magnus Aronsen, Ida Gjervold Lunde, Bjørn Dalhus, Marianne Lunde, Heidi Kvaløy, William Edward Louch, Theis Tønnessen, Ivar Sjaastad, Ole Mathias Sejersted and Cathrine Rein Carlson. J Biol Chem. 2014 Dec 5; 289(49):33984-98.

2. Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman Tandekile Lubelwana Hafver, Kjetil Hodne, Pimthanya Wanichawan, Jan Magnus Aronsen, Bjørn Dalhus, Per Kristian Lunde, Marianne Lunde, Marita Martinsen, Ulla Helene Enger, William Fuller, Ivar Sjaastad, William Edward Louch, Ole Mathias Sejersted and Cathrine Rein Carlson.J Biol Chem. 2016 Feb 26; 291(9):4561-79.

3. Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1)

Pimthanya Wanichawan, Kjetil Hodne, Tandekile Lubelwana Hafver, Marianne Lunde, Marita Martinsen, William Edward Louch, Ole Mathias Sejersted and Cathrine Rein Carlson.

Biochem J. 2016 Aug 1; 473(15):2413-23.

4. Mapping the in vitrointeractome of cardiac Na⁺-Ca²⁺exchanger 1 (NCX1)

Tandekile Lubelwana Hafver, Gustavo Antonio de Souza, Pimthanya Wanichawan, Marianne Lunde, Marita Martinsen, Ole Mathias Sejersted and Cathrine Rein Carlson.

Manuscript in revision. PROTEOMICS-pmic.201600417

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1 Introduction

1.1 Brief historical perspective

The function of the heart is a topic that has interested scholars since antiquity, but William Harvey (1578-1657) is commonly cited as the pioneer of modern cardiovascular research. Harvey challenged previous beliefs that blood is continuously produced from digested food and consumed by the organs in the body, and postulated instead that the blood flows in a cycle, and that the heart’s function is to pump the blood through the circulatory system (1). Harvey’s theories soon gained acceptance, but another question remained a puzzle: What drives the heartbeat?

Over the next centuries, two competing theories emerged: On the one hand, the myogenic theory held that excitation originates in the heart itself. On the other hand, the neurogenic theory proposed that excitation originates from nerve connections or ganglions. This debate was eventually settled in favor of the myogenic theory: Walter Gaskell (1847–1914) observed that an isolated strip of tortoise ventricular muscle, devoid of ganglions or nervous connections, continued to pulsate at a rate similar to the intact heart, and concluded that “The rhythmic capacity of every part of the heart depends not upon the presence of ganglion cells but rather upon the persistence of a primitive condition of heart muscle”(2). Consequently, through a sequence of discoveries by, among others, Jan Evangelista Purkinje (1787–1869), Wilhelm His Jr (1863–1934) and Sunao Tawara (1873–1952) (3), a network of electrically conducting pathways in the heart was revealed. In 1907, Arthur Keith and Martin Flack identified the sinus node, which contains special pacemaker cells that produce and transmit the electrical impulses that drive the heartbeat, the so called action potential (4).

The next big question was how the action potential translates into contraction of the heart muscle. The first hints had been provided by Sydney Ringer, in a series of four papers in the 1880s (5- 8). Ringer demonstrated that the force of contraction of frog hearts was influenced by the concentrations of various ions in the perfusion solution he used in his experiments. Crucially, he discovered that the isolated frog hearts could not be made to contract in the absence of Ca²⁺. Thus, Ca²⁺was identified as a crucial mediator of contraction.

In 1948, Willibrandt and Koller (9) reported that cardiac contraction increased in the presence of low concentrations of Na⁺. The importance of Na⁺for contractility was further recognized when, in 1953, Schatzmann discovered that cardiac glycosides (such as digitalis, or foxglove, which had been used for centuries as a remedy for treating heart conditions (10)) work by inhibiting K⁺ and Na⁺ transport (11). In 1958, Lüttgau and Niedergerke reported that the force of contraction in fact depended on the ratio of extracellular Ca²⁺and Na⁺(12,13). A decade later, Reuter and Seitz (1968) and Baker and Blaustein et al. (1969), through experiments on guinea pig atria (14) and squid giant

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axons (15) respectively, were the first to show a coupling between influx and efflux of Ca²⁺and Na⁺ across the plasma membrane and documented the existence of a Na⁺-Ca²⁺ exchange counter-transport system. This coupling between Na⁺and Ca²⁺transport explained how the blocking of Na⁺transport by cardiac glycosides could regulate contractility by affecting the Ca²⁺loading of cardiac cells (16).

Over the past half century, the role of Ca²⁺and Na⁺transport in cardiac excitation-contraction coupling has been further characterized and clarified, but it still remains an active focus of research in elucidating disease mechanisms.

1.2 The sodium–calcium exchanger 1 (NCX1)

The sodium–calcium exchanger (NCX) is a ubiquitously expressed ion-transporting plasma membrane protein that plays an important role in maintaining cytosolic Ca²⁺homeostasis. Splice variant NCX 1.1 (referred to as NCX1 in this thesis) is cardiac specific, and plays an important role in excitation- contraction coupling in cardiomyocytes as it is the dominant cellular Ca²⁺ efflux mechanism mediating diastolic relaxation and preparing the cell for the next contraction cycle (17).

The physiological importance of NCX1 in early development is illustrated by the observation that ubiquitous deletion of NCX1 brings about early death of embryos because of a lack of heartbeat (18). Studies have also reported increased NCX1 messenger RNA (mRNA) and protein levels in end- stage human heart failure (HF) (19,20), but, interestingly, this is not always correlated with increased NCX1 activity (21). Such observations have elicited interest in NCX1’s role in pathophysiological conditions and as a potential therapeutic target (21). Two features which makes NCX1 particularly interesting in this regard are the facts that it can exchange Ca²⁺ for Na⁺in both directions, and that it operates at a stoichiometry of one Ca²⁺exchanged for three Na⁺’s, making its transport electrogenic.

This means that NCX1 can play a role both in Ca²⁺import and removal, and by carrying charge, it can also influence the configuration of the action potential and contributing to arrhythmias (22). However, if NCX1 is to be a viable therapeutic target, a better understanding of its regulation is warranted, motivating the work contained in this thesis.

1.2.1 Molecular biology of NCX1 and splice variants of NCX

An important step in the study of NCX1 was the successful purification of NCX1 in 1988 (23). The isolation of the exchanger paved the way for the production of polyclonal antibodies which were used in the cloning of NCX1 from canine hearts in 1990 (24). This has allowed subsequent studies of the exchanger at the molecular level.

The initial molecular cloning of NCX1 was followed by the identification of several related proteins through sequence database mining and low-stringency library screening. It was established that NCX1 is a member of a larger gene family, the solute carrier (SLC) 8 family, which, in turn, forms part of the Ca²⁺/cation (CaCA) antiporter superfamily (25,26). Three gene members of the SLC8

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family are expressed in mammals: SLC8A1 (NCX1) (24), SLC8A2 (NCX2) (27), and SLC8A3 (NCX3) (28). These give rise to at least 17 NCX1 and 5 NCX3 alternative splice variants, but no splice variants of NCX2 are known. The splice variants can be attributed to six small exons (A, B, C, D, E, and F), of which exons A and B are mutually exclusive and appear in every splice-variant for NCX1 and NCX3, resulting in tissue specific expression (29). Although first discovered in cardiac cells, NCX1 is the most broadly expressed SLC8 family member, present at low levels in most tissues, and expressed at high levels in the brain and kidneys, in addition to the heart (26). The three mammalian SLC8 family members share about 70% amino acid identity overall, and more than 80% identity within the predicted transmembrane segments (30). The latter indicates a common function of ion exchange.

1.2.2 Topology and structure of NCX1

Human NCX1 is 973 amino acids long, with the first 32 amino acids representing a signaling peptide that is cleaved off during processing. This signal sequence was originally believed to ensure the correct insertion of the exchanger into the cell membrane, however, the exchanger is correctly targeted even when this sequence is removed (31). The N-terminus of the protein is glycosylated, which does not affect exchanger function, but helps to define the exchanger topology (32). On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the NCX1 protein displays three bands with apparent molecular masses of 70, 120, and 140 kilodalton (kDa). The 120-140 kDa band corresponds to the full-length mature protein, whereas the ~70 kDa band represents a proteolytic fragment (23). The 120-140 kDa full-length doublet is attributed to a mobility shift due to the formation of an intramolecular disulfide bond between Cys-792 and either Cys-14 or Cys-20 (33).

Additionally, intermolecular cross-linking (34) and fluorescence resonance energy transfer (FRET) (35) suggests that NCX1 forms dimers. It has even been suggested that NCX1 exists as a higher-level multimer in the membrane (36). The physiological implication of these NCX1 multimers remains unknown.

Members of the SLC8 family are characterized by a conserved overall membrane topology, with two clusters of hydrophobic transmembrane (TM) domains, joined by a cytosolic loop whose length varies between the family members (26). Based on accessibility experiments and epitope mapping, the current model for mammalian NCX1 has nine TM domains; five in the first hydrophobic cluster and four in the second (37,38). This has been challenged, however, by recent studies reporting that the crystal structure of a prokaryotic homologue contains 10 TM domains (39). There is strong precedent for prokaryotic and eukaryotic homologues of membrane proteins to have a similar topology (40).

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The conserved hydrophobic TM domains of NCX1 (residues 1–217 and 727–903) (41) contain WZRLQWHUQDOO\KRPRORJRXVUHJLRQVWHUPHGWKHĮ-DQGĮ-UHSHDWV7KHĮ-repeats are believed to have arisen from an ancient gene-duplication event (42)DQGPXWDJHQHVLVVWXGLHVKDYHLQGLFDWHGWKDWWKHĮ- repeat regions catalyze ion translocation (43). Crosslinking experiments have shown that the helices predicted to flank the Į-repeats are in close proximity (44,45), and accessibility experiments have indicated that the two Į-repeats are oriented in opposite directions with respect to the membrane, possibly with their central regions forming membrane-reentrant loops with limited accessibility from either side of the membrane (37,38). More than half of the NCX1 protein is made up of a ~ 550 amino acid hydrophilic cytosolic loop between TM5 and TM6. Deletion of this cytosolic loop does not affect ion translocation, however, the loop mediates regulation of the exchanger by associating with various cytosolic factors (46-51). The cytosolic loop of NCX1 can be broken down into different domains: 1) The N-terminal exchanger inhibitory peptide (XIP) domain (residues 219–238 ), which binds calmodulin (31). 2) The catenine-like domain (CLD) (residues 218–370 and 651–726) (41), which contains binding sites for phospholemman (49), a calpain cleavage site (paper 1) and an endogenous XIP site (52). 3) Regulation sites for two Ca²⁺-binding domains (CBD), CBD1 (residues 371–500) and CBD2 (residues 501–650) (41), where Ca²⁺ binding alters the structure and motional dynamics of these domains (41,53-55). Recently, CBD1 has been shown by us to have anchoring sites for calpain (paper 1) (48) and protein phosphatase 1 catalytic domain (PP1c) (paper 2) (50). The amino acid numbering used above excludes the signaling peptide sequence. The nuclear magnetic resonance 105VWUXFWXUHVRIWKH&%'VUHYHDOWKDWWKH\KDYHWKHDUFKLWHFWXUHRIDȕ-sandwich, formed by two DQWLSDUDOOHOȕ-sheets (41) (Fig. 1). The structures of CBD1 and CBD2 have been elucidated, but the three-dimensional structures of the CLD remain unsolved.

Figure 1. Predicted topology of NCX1. The figure shows that NCX1 is composed of 10 TM domains, FRQWDLQLQJĮDQGĮUHSHDWVDQGDODUJHF\WRVROLFORRSFRQWDLQLQJWKH;,3&/'&%'DQG&%'GRPDLQV The CBD1 and CBD2 are arranged as anti-SDUDOOHO ȕ-sheets and bind Ca²⁺with high affinity. Figure obtained from (56) and modified to show the presence of 10 TM domains. Reprinted with permission from the publisher:

Portland Press Ltd.

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1.2.3 Function

NCX1 plays an important role in maintaining intracellular Ca²⁺ homeostasis, as it is the primary contributor to Ca²⁺efflux. In the heart, NCX1 mediates diastolic relaxation by transporting Ca²⁺across the plasma membrane in exchange for Na⁺, at a stoichiometry 1:3. The exchanger can operate in both Ca²⁺efflux (forward mode) and Ca²⁺ influx (reverse mode), depending on the internal and external concentration of both Na⁺and Ca²⁺, as well as on the membrane potential (57).

1.2.3.1 NCX1 role in excitation-contraction coupling

The mechanical contraction of the heart is linked to the electrical action potential through Ca²⁺, as illustrated in Fig. 2. In brief, the ventricular action potential begins with the depolarization of the cell membrane, caused by an electrical impulse transmitted from a neighboring cardiomyocyte. This causes voltage gated Na⁺channels to open and Na⁺ to enter the cell, raising the membrane potential.

As the membrane potential increases, voltage gated L-type Ca²⁺ channels (LTCCs) are activated, allowing extracellular Ca²⁺to flow down their Ca²⁺gradient into the cytosol. Additional Ca²⁺may also enter the cell via NCX1 operating in reverse mode (17,58). Cytosolic Ca²⁺ ions diffuses across the dyadic cleft, and binds to clusters of ryanodine receptors (RYR2) localized on the membrane of the sarcoplasmic reticulum (SR). This triggers the RYR2 to open, and Ca²⁺ is released from the SR in a series of sparks, a process called Ca²⁺induced Ca²⁺release (CICR) (59). This process rapidly increases cytosolic Ca²⁺ concentrations and Ca²⁺ binds to Troponin C of the myofilaments and induces a conformational change that induces the interaction between actin and myosin which allows cross- bridges to form, resulting in contractile force generation. In order for the cardiomyocyte to relax and prepare for the next contraction cycle, Ca²⁺must be removed from the cytosol. Most of the Ca²⁺is re- sequestered into the SR, mediated by sarcoendoplasmic reticulum Ca²⁺ATPase 2 (SERCA2), while a small amount of Ca²⁺is taken up by the mitochondria and plasma membrane Ca²⁺ ATPase (PMCA).

The rest of the Ca²⁺is extruded from the cell, mainly via NCX1. Similarly, Na⁺balance is restored by the sodium/potassium-transporting ATPase (NKA) (Fig. 2).

Note that NCX1 plays several roles in the excitation-contraction coupling cycle: Firstly, it serves as the primary Ca²⁺ extrusion mechanism during diastole. Secondly, the contribution from reverse mode operation of NCX1 in systole, although small, may prime dyadic RYR2 in its vicinity for CICR. Furthermore, as its transport is electrogenic (carries net charge), NCX1 also modulate the action potential.

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Figure 2. Schematic figure of excitation-contraction coupling in ventricular cardiomyocyte. The figure shows the main participating proteins in excitation-contraction coupling, as described in the text above. The inset shows the progression of the action potential and Ca²⁺ transient, which drives the contraction. Figure obtained from (17). Reprinted with permission from the publisher: Nature Publishing Group.

1.2.3.2 NCX1 in pathophysiology

The various ion channels in the heart participate in complex interplay, and the correct balance between different ion transport mechanisms is important to ensure proper functioning of the heart. Improper handling of Ca²⁺is a common cause of both contractile dysfunction and arrhythmias in HF.

Increased NCX1 mRNA and protein levels have been shown in end-stage human HF (19,20), and elevated activity of NCX1 has been linked to dysfunctional Ca²⁺handling in chronic heart disease (29). Overexpression of NCX1 in HF can lead to increased Ca²⁺efflux through forward mode NCX1 activity, which combined with SERCA2 dysfunction can lead to depletion of SR Ca²⁺ and result in reduced contractile force (60). Abnormal efflux of Ca²⁺during diastole could also lead to delayed afterdepolarizations and contribute to arrhythmias. In addition, increased reverse mode NCX1 activity can also contribute to the triggering of spontaneous and unsynchronized Ca²⁺ release from the SR (61,62).

Insights regarding NCX1 function and the therapeutic potential of targeting the exchanger can be learned from transgenic mouse models. Global knock-out of NCX1 is embryonically lethal (18) due to a lack of heartbeat, indicating a crucial function of NCX1 in early development. Surprisingly, however, mice with conditional knock-out of NCX1 from the ventricle myocytes survive, with only 20-30% reduction in contractility (63). The survival can be attributed to two adaptations (64): Firstly, reduced NCX1 Ca²⁺ efflux is compensated by a ~80% reduction of Ca²⁺influx via LTCC, and Ca²⁺

expulsion is mediated by PMCA, while the SR Ca²⁺is not affected (65). Secondly, the action potential is shortened, which further reduces the inflow of Ca²⁺(64). These adaptations serve to maintain both

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cytosolic Ca²⁺homeostasis and contractility. In summary, the knock-out of NCX1 represents a gain in contractile efficiency (since less Ca²⁺ influx is required to maintain contraction), but at the cost of depressed Ca²⁺transient amplitude, elevated diastolic Ca²⁺concentration and shorter action potentials (64). Similarly to the conditional knock-out mice, NCX1 overexpressing mice also display almost normal cardiac function, but with opposite adaptations: Increased Ca²⁺ extrusion via NCX1 is compensated by increased Ca²⁺influx via LTCC, and the mice exhibit smaller Ca²⁺transients and a prolonged action potential (66).

Whether up-regulation of NCX1 during cardiac hypertrophy and HF is adaptive or maladaptive is not fully understood, not least because the heart function and the remodeling that occurs during HF involves a multitude of proteins interacting in complex ways. In HF, increased NCX1 activity could contribute to arrhythmia, as well as Ca²⁺ depletion of the SR and reduced contractility, but, on the other hand, this may also protect against diastolic Ca²⁺overload (67).

Inhibition of NCX1 was reported to be protective against cardiac ischemia–reperfusion injury (68), which may be explained by reduced Ca²⁺entry via NCX1.

1.2.4 Regulation

1.2.4.1 Regulation by ions

The most prominent regulatory mechanisms of NCX1 are induced by Na⁺and Ca²⁺, which, in addition to being the substrates for transport, also have separate regulatory roles. On the one hand, binding of intracellular Ca²⁺to CBD1 and CBD2 induce conformational changes that causes allosteric activation of the exchanger, as first observed by DiPolo in 1979 (69). Activation occurs in the physiological range, just above resting Ca²⁺ level, 150–400 nM (70). Ottolia et al. 2004 showed in FRET experiments that the Ca²⁺activation of the exchanger is fast enough to regulate NCX1 on and off on a beat to-beat basis (71). Ca²⁺ gating could be a mechanism to ensure that cytosolic Ca²⁺ does not decline too much between beats. On the other hand, Na⁺ inactivates the outward exchange current when applied to the intracellular surface of the cell membrane (72,73). This mechanism involves the XIP region in the cytosolic loop of NCX1. Mutations in the XIP region alters the susceptibility of the exchanger to inactivation by Na⁺, where the F223E mutant makes the exchanger hypersensitive, while the K229Q mutant makes the exchanger resistant to Na⁺ inactivation (74). Binding of phosphatidylinositol-4,5-bisphosphate (PIP₂) to the XIP region disrupts the Na⁺-dependent inactivation, and adenosine triphosphate (ATP), which stimulates synthesis of PIP2 from phosphatidylinositol (PI), can therefore protect against inactivation of the exchanger (75). A rise in cytosolic Ca²⁺, on the other hand, relieves the Na⁺-dependent inactivation (76). Na⁺-inactivation is not believed to be important on a beat-to-beat basis or under normal physiological conditions, as the wild type exchanger only responded to Na⁺-dependent inactivation when the cytosol was acidified (73). It is instead believed that this mechanism might play a protective role in conditions of high pH and high Na⁺, such as in

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ischemia, by inhibiting NCX1 mediated Ca²⁺influx (77). In addition, NCX1 is also regulated by other divalent and trivalent cations which have been shown to inhibit Ca²⁺ transport by the exchanger (78,79).

1.2.4.2 Regulation by calpain

Proteases are enzymes that catalyze the cleavage of peptide bonds by hydrolysis. Calpains are a family of non-lysosomal calcium-dependent cysteine proteases, whose enzymatic effect is to cleave its substrates in response to high Ca²⁺ concentrations. Calpain is often referred to as a ‘modulator protease’, because, unlike lysosomal proteases, it does not digest its substrates. Rather, calpain proteolysis is selective and is limited to specific sites, modulating the function or activity of its substrates (80).

The nomenclature for calpains has evolved with the discovery of new members. It is common to classify calpains into classical/conventional/typical and non-classical/unconventional/atypical calpains, where the former includes calpain-1 and calpain-2 (81). The classical calpains are ubiquitously expressed heterodimeric proteins with two distinct subunits; 1) a large catalytic subunit (~80 kDa) and 2) a small regulatory subunit (~28 kDa). The regulatory subunit is common for calpain- 1 and calpain-2, whereas their catalytic subunits are similar, but distinct, with 55-65% sequence homology (80,82). On SDS-PAGE calpain migrates as a doublet, as autolysis reduces the mass of calpain-1 to 76 kDa and calpain-2 to 78 kDa (83). The domain structure of the catalytic subunit of calpain-1 and calpain-2, as well as the regulatory subunit, is shown in Fig. 3.

An important regulator of calpain activity is Ca²⁺. Calpains undergo conformational changes in response to Ca²⁺ binding, causing the alignment of the active site. The classical calpain isoforms differ in their Ca²⁺activation thresholds: Calpain-1 has a half-maximal activity at 3-ȝ0&D²⁺, and is often called micro-FDOSDLQȝ-calpain). Calpain-2 has a half-maximal activity at 400-ȝ0&D²⁺, and is often called milli-calpain (m-calpain) (84). The Ca²⁺ concentrations required to activate both calpain-1 and calpain-2 is significantly higher than resting concentrations in cells. This means that calpain activation only can occur locally near influx points of Ca²⁺, and the activation is temporarily limited by diffusion and removal of Ca²⁺ by ion pumps (85). Calpain activity is also regulated by an endogenous inhibitor, calpastatin, which inhibits calpain activation, preventing unspecific activation by normal Ca²⁺ flux. In the presence of excess Ca²⁺ levels, calpastatin will dissociate and allow calpains to autolyse and become an active protease against various substrates (86). The small regulatory subunit of calpain mediates its regulation by ensuring proper folding of the large catalytic subunit and is also essential for the proteolytic activity of calpain-1 and calpain-2 (87).

Through proteolytic processing, calpains affect a range of cellular processes by modifying its substrates. Although the exact functions of calpain remain elusive, it is linked to signal transduction, cell cycle progression, regulation of gene expression, apoptosis (80) and cleavage of enzymes, and

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cytoskeletal/membrane proteins (88). Calpain has also been implicated in various pathologies.

Specifically, activation of calpain-1 has been shown to cause destruction of contractile filaments in fibrillating atrial muscle (89), and increased calpain activity is reported to exacerbate pathologies relating to lack of Ca²⁺ control (84). Inhibition of calpain at the onset of reperfusion was shown to have cardioprotective effects (90). Insights into the function of calpain may be gained from studies in transgenic mouse models. Calpain-1 knock-out mice are viable and fertile, with some defects in platelet aggregation (91), while calpain-2 knock-out mice exhibit embryonic lethality. This suggests a crucial role of calpains, but suggests that calpain-2 may be able to compensate for calpain-1. However, knockout of the regulatory subunit in both calpain-1 and calpain-2 is embryonically lethal (92), indicating an important role of the regulatory subunit as an endogenous regulator of calpain activity.

Figure 3. Domain structure of calpains. In the catalytic subunit, the N-terminal anchor helix region is designated as domain I. Domain II contains the Cys, His, and Asn amino acid residues necessary for proteolytic function. Domain III consists of residues that associate with plasma membranes and binding Ca²⁺. Domain IV contains five EF hand sequences responsible for Ca²⁺ binding. The small regulatory subunit (CAPNS) is common to both μ-calpain (calpain-1) and m-calpain (calpain-2) isoforms and is composed of Domains V and VI. These domains contain hydrophobic residues that help to achieve membrane interaction and additional Ca²⁺

binding. The catalytic and regulatory subunits remain associated under inactive conditions. Upon Ca²⁺ binding, μ-calpain (calpain-1) and m-calpain (calpain-2) self-cleave an N-terminal portion of the large and small subunits to achieve full proteolytic activity. Figure obtained from (82). Reprinted with permission from the publisher:

John Wiley and Sons. Of note, a new nomenclature was proposed for the domains and is summarized in (93).

As mentioned above, calpain mediates proteolysis of cytoskeletal/membrane proteins, and NCX1 has been identified as a substrate for calpain in the brain (94). However, the molecular mechanisms regulating calpain cleavage of NCX1 in the heart remained unknown. In paper 1 of this thesis we have shown that treatment of left ventricle (LV) tissue lysates with calpain resulted in a ~75 kDa proteolytic fragment (95). Further, we mapped the calpain binding and cleavage site in NCX1 and investigated the functional consequence of NCX1 cleavage in human embryonic kidney 293 (HEK293) cells.

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1.2.4.3 Regulation by protein phosphatase 1 (PP1)

The balance between the activity of protein kinases and phosphatases regulates diverse physiological processes, such as cell division, cell differentiation, neuronal activity, muscle contraction and metabolic functions (96). 3% of all human genes encode protein kinases or phosphatases, indicating their importance (97).

Protein phosphatase 1 (PP1) is a ubiquitously expressed ~38.5 kDa serine/threonine phosphatase which counters the effects of serine/threonine kinases (98). Mammalian genomes encode IRXUGLVWLQFWFDWDO\WLFVXEXQLWVRI3333Į33ȕ»įDQGWKHVSOLFHYDULDQWV33FȖDQG33FȖ(99).

The isoforms show 85% sequence identity, but the N- and C-terminal extremities show amino acid differences, allowing for distinct tissue distribution and subcellular localization (99).

The first crystal structure of the PP1-microcystin complex was shown by Goldberg et al. 1995 and revealed that the PP1 catalytic domain (residues 7-KDVDȕ-Į-ȕ-Į-ȕVFDIIROGLQJZLWKDVKDOORZ active site (100). This active site contains two metal ions which catalyze the dephosphorylation reaction. Additionally, the active site is surrounded by various substrate-binding grooves, which increase the number of possible substrates recognized by PP1, as shown by the surface representation in Fig. 4. The catalytic domain (PP1c) achieves substrate specificity by forming holoenzymes with more than 200 regulatory proteins (101). These regulatory proteins localize PP1c to specific subcellular domains and fine-tune its activity, allowing for substrate specific effects (101). Regulatory proteins can affect the activity of PP1 by serving as inhibitors, pseudosubstrate, substrates or substrate- specifiers (102). PP1 activity is regulated by toxins such as microcystin (103) and okadaic acid (104), which inhibit PP1 activity potently, but not specifically. PP1 is also inhibited by protein inhibitors, such as inhibitor-1 (I-1), dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32,000 (DARPP-32) and inhibitor-2 (I-2). I-1 and DARPP-32 must first be phosphorylated by protein kinase A (PKA) in order to convert them into potent inhibitors.

90% of PP1 regulatory proteins interact with PP1 via a short degenerate RVxF-docking motif with consensus sequence [RK]x_0-1[VI]{P}[FW], where x denotes any residue and {P} any residue except proline (105). The RVxF motif serves as an anchor for the initial recruitment of PP1 and enables the subsequent establishment of secondary PP1 interactions. Additional recently identified docking motifs have been described, such as the SILK and MyPhoNE motifs found in six of the 200 known PP1 regulator proteins. The former anchors PP1, while the latter is important for substrate selection (106,107). Choy et al. 2014 defined an extended PP1 binding motif with consensus sequence RVxF-X_5–8-ĭĭ-X_8–9-R, where the RVxF motif is followed by a variable, two-residue phi-phi ĭĭPRWLIDQGDFRQVHUYHGDUJLQLQH5(108). The conserved arginine in the PP1 nuclear targeting subunit (PNUTS)-PP1 holoenzyme has been shown to work as a substrate selectivity filter, which blocks access to the C-terminal binding groove in PP1 (108).

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Figure 4. The structure of PP1 from the PP1-microcystin complex is shown as a surface representation.A:

7KHVKDOORZDFWLYHVLWHRI33ĮJUHHQFRQWDLQVWZRPHWDOLRQV0Q²⁺, but likely Fe²⁺and Zn²⁺in vivo) (103) (pink spheres), which catalyzes the dephosphorylation reaction and lies at the Y-shaped intersection of three substrate-binding grooves; the hydrophobic (blue), the acidic (orange), and the C-terminal (red). B: The rotation of (A) shows the binding sites for the PP1-docking motifs RVxF (purple), SILK (cyan) and MyPhoNE (wheat) (PDB ID 1FJM). Figure obtained from (101). Reprinted with permission from the publisher: Elsevier Ltd.

The physiological relevance of PP1 in the heart was shown by the use of okadaic acid, a nonspecific PP1 inhibitor, which enhanced contractility in guinea-pig isolated ventricular muscles (109).

7UDQVJHQLF PRXVH PRGHOV ZKLFK RYHUH[SUHVV WKH 33Į FDWDO\WLF VXEXQLW GHYHORSHG +) VKRZLQJ impaired cardiac contractility, ventricular dilatation, increased cardiac fibrosis and re-expression of fetal genes (110). Overexpression of constitutively active forms of I-1 (111) or I-2 (112) shows the opposite phenotype as in overexpression of PP1. Active I-1 and I-2 lowered PP1 activity, restored adrenergic responsiveness, enhanced cardiac contractility and is protective against the development of cardiac hypertrophy.

PP1 mediates regulation in cardiac electrophysiology and in key contractile proteins by controlling their dephosphorylation after phosphorylation by protein kinases. PP1 has been shown to target the K⁺ channel macromolecular complex composed of the pore-IRUPLQJ Į VXEXQLW DQG PRGXODWRU\ȕVXbunit K⁺FKDQQHO3.$DQG$.$37KLVLQWHUDFWLRQPHGLDWHGȕ-adrenergic signaling (113). RYR2 is phosphorylated on Ser-2030 and Ser-2808 by PKA (114) whereas calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Ser-2814 (115).

Dephosphorylation of RYR2 is mediated by PP1, which is targeted to the complex by the regulatory protein spinophilin. The absence of spinophilin decreases PP1-mediated dephosphorylation of RYR2 and leads to RYR2 hyperactivity, contributing to atrial fibrillation (116). PP1 also mediates regulation of phospholamban (PLN), the accessory protein which regulates the activity of SERCA2 (117,118).

Dephosphorylation of phospho serine-68-phospholemman (pSer-68-PLM) by PP1 was recently shown to modulate phospholemman (PLM) regulation of NKA (119). pSer-68-PLM also mediates regulation of NCX1 (see section 1.2.4.4), but it is unknown whether PP1 is involved in NCX1 regulation. In paper 2 of this thesis we investigated whether a direct and functional NCX1-PP1c interaction is a prerequisite for dephosphorylation of the pool of pSer-68-PLM which regulates NCX1.

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1.2.4.4 Regulation by phospholemman (PLM)

PLM is a sarcolemmal phospho protein that was purified and cloned in 1991 (120). Early studies on PLM showed that it is an anion-selective channel, which regulates cell volume in noncardiac tissues (121). Later studies showed that it is part of the PFXYD family (named after a conserved Pro-Phe-X- Tyr-Asp motif in the extracellular N-terminus domain) of regulators of ion transport, and this extended the role of PLM to excitable tissue (122). PLM has been shown to be expressed in the heart, skeletal muscle, brain, liver and kidneys.

PLM, also known as FXYD1, is synthesized as a 92 amino acid peptide, with the first 20 amino acids being a signal peptide sequence that is cleaved off during processing to form the mature protein containing 72 amino acid residues. The topology of the mature protein shows that it is composed of the signature PFXYD motif, which is located in the extracellular N-terminus and is resistant to protease degradation, a single hydrophobic transmembrane domain and a cytosolic tail (120). The cloned complementary deoxyribonucleic acid (cDNA) of PLM, from cardiac muscle, revealed that it has a molecular weight of 8409 Dalton. On SDS-PAGE, however, it migrates with molecular weight 15-kDa, due to the slow migration of the PLM transmembrane domain (123). In human and rat, the cytosolic tail of PLM contains three serines (at residues 62, 63, and 68) and one threonine (at residue 69) (Fig. 5A), but Thr-69 is replaced by serine in mouse (122). NMR showed that WKH 3/0 PROHFXOH FRQVLVWV RI IRXU Į-helices which are rigidly connected, where helix 3 and 4 are connected by a flexible linker (Fig. 5B) (124). PKA phosphorylates Ser-68, whereas protein kinase C (PKC) phosphorylates Ser-63 and Ser-68 of PLM (125).In vitrostudies suggest that Ser-63 and Thr- 69 may be additional phosphorylation targets for PKA and PKC, respectively (126). Phosphorylation of Ser-68-PLM has been shown to enhance palmitoylation at Cys-40 and Cys-42, leading to NKA inhibition (127,128). Cys-42 can also be glutathionylated (129). The basal phosphorylation level of PLM, by endogenous kinases, has been reported to be 30–40% in adult rat myocytes and HEK293 (47,130).

PLM is proposed as a novel cardiac stress protein which modulates myocardial contractility by regulating the activity of proteins involved in excitation-contraction coupling. PLM regulates the activities of LTCC (131), NKA (132) and NCX1 (130) in mammalian hearts. PLM modulates Ca²⁺

entry through LTCC in adult LV myocytes by reducing peak LTCC current amplitude and enhancing voltage-dependent inactivation. Only the transmembrane and the extracellular (signature PFXYD motif) domains of PLM mediates the regulation of LTCC (133). Unphosphorylated PLM exerts inhibition on NKA, whereas phosphorylation relieves this inhibition (132). It is PLM’s transmembrane domain (Phe-28) which interacts with TM9 (Glu- RI WKH Į-subunit of NKA (134) and phosphorylation alters the PLM-NKA interaction, but does not mediate disassociation of PLM from NKA (135). PLM overexpression by adenovirus-mediated gene transfer decreased NKA current in adult rat myocytes (136), whereas PLM knock-out mice expressing the phosphomimetic PLM Ser-68-

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Glu (S68E) mutant inhibited NCX1 current without any effect on NKA activity (137).

Unphosphorylatable PLM reduces NKA activity and exacerbates Na⁺overload, resulting in contractile dysfunction and adverse remodeling following aortic constriction in mice (138).

Figure 5. PLM domains and structure.A: PLM domain organization and human sequence is shown. PLM is composed of a signal peptide sequence (in blue) that is cleaved off during protein processing; the extracellular domain which has the signature FXYD motif (in bold red) which is located in the extracellular N-terminus; a single hydrophobic transmembrane domain (in grey) and a cytosolic tail with the palmitoylation site at Cys-40 and Cys-42, glutathionylated sites at Cys-42 and phosphorylation sites at Ser-63, Ser-68 and Thr-69 (in bold). B:

Ribbon diagram of PLMVKRZVWKDWLWFRQVLVWVRIIRXUĮ-helices which are rigidly connected where helix 3 and 4 are connected by a flexible linker (PDB ID: 2JO1).

The role of PLM in NCX1 regulation was first described by Song et al. 2002, where they found that overexpression of PLM altered Ca²⁺transients and reduced contractility (136). This led to speculations that PLM directly regulates NCX1 independently of NKA (136). Ahlers et al. 2005 identified PLM as an endogenous inhibitor of cardiac NCX1 (139), specifically that it is the phosphorylation of PLM at Ser-68 which mediates this inhibition (125,130). The PLM binding region on NCX1 was constrained to the PASKT (residues 248–252) and QKHPD (residues 300–304) containing sequences, and mutating these binding regions resulted in loss of PLM induced NCX1 inhibition in HEK293 (49). The possibility that PLM indirectly regulates NCX1 via Ca²⁺was excluded by the fact that NCX1 lacking both CBD1 and CBD2, expressed in HEK293, were still inhibited by PLM (140). Upon examination of the now known PLM binding region in NCX1, this finding is explained by the fact that the PLM anchoring site on NCX1 is outside the CBDs. The physiological significance of PLM is shown under stress conditions (141): Relieved inhibition of NKA by pSer-68- PLM can prevent Ca²⁺ and Na⁺ overload, which can reduce the risk of arrhytmogenesis. The latter reduces contractility, but this is counteracted by simultaneous inhibition NCX1. The PLM-NCX1 mechanism can therefore serve to improve or maintain contractility under stress conditions.

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In paper 3 of this thesis we designed a high-affinity peptide for PLM, which is derived from NCX1, and investigated its role in disrupting the PLM-NCX1 interaction.

1.2.4.5 Regulation by other cytosolic factors

Various cytosolic factors interact with the cytosolic loop of NCX1 and mediate regulation of the exchanger. We only mention the factors where the interaction site in NCX1 has been mapped down to amino acid level. Annexin V, has been shown to interact with the Ca²⁺-activation site (371-525) in NCX1, where this interaction locally decreases the amount of Ca²⁺ available for NCX1 (142,143).

Calmodulin-1-3 interacts with the XIP domain (251-270) (31) and inhibits NCX1 activity (31). Further, calmodulin-1-3 binds to the 1-5-8-14 calmodulin binding segment motif in NCX1 (716–735) (144) and releases the auto-inhibitory domain which enhances NCX1 transport activity. Caveolin has been shown to interact with the endogenous XIP site in NCX1 (145) and forms part of a macromolecular complex with annexin A5. Creatine kinase M-and S-type interact with NCX1 (231-381) and NCX1 (226–380) respectively, and this interaction recovers reverse-mode NCX1 activity under energy- compromised conditions (146). As mentioned above (section 1.2.4.4), PLM interacts with PASKT (248-252) and/or QKHPD (300-304) in NCX (49), and phosphorylation of Ser-68-PLM inhibits NCX1 activity (125,139,141). Calcineurin interacts with NCX1 (407–478) and decreases NCX1 activity (147). NCX1 XIP region also interacts with phospholipid, PIP₂, which activates NCX1, as PIP₂ eliminates the Na⁺-dependent inactivation (148). Phosphorylation of NCX by different protein kinases, such PKA (149) and PKC (150) has been demonstrated in the heart to increase NCX1 activity, however other laboratories have failed to see these kinase dependent effects (42,151). Our laboratory has shown that the PKA phosphorylation site in NCX1 is inaccessible for phosphorylationin vitro(95).

Cys-739 in NCX1 has been shown to be palmitoylated in cardiac muscle, and it is suggested that palmitoylation is required for the XIP domain to fully inhibit NCX1 exchange activity. This shows that palmitoylation regulates NCX1 inactivation (152). In paper 4 of this thesis we used a proteomic approach to screen for novel cytosolic factor which may interact with NCX1 and mediate its regulation.

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2 Aims of the thesis

NCX1 participates in numerous physiological and pathophysiological processes. Altered expression, regulation and activity of NCX1 contributes to distorted Ca²⁺homeostasis in cardiomyocytes affecting excitation-contraction coupling. There are no selective NCX1 modulators that are in clinical use, and pharmacological targeting of NCX1 has been desired for a long time. In this thesis we identify and validate novel NCX1 interacting partners. We elucidate the mechanism of regulation and design a new pro-drug.

Paper 1

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

NCX1 has previously been identified as a substrate for Ca²⁺ activated proteases such as calpains, however, little was known about the precise molecular mechanisms and biological consequences of calpain-dependent cleavage of NCX1. In this study we hypothesized that calpain is an important regulator of NCX1 in response to pressure overload.We aimed to identify the molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in conditions of hypertrophy and HF relative to normal heart.

Paper 2

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

Direct regulation of NCX1 by kinases is controversial; instead it has been suggested that phosphorylation and dephosphorylation occurs on accessory proteins in the NCX1 macromolecular complex. PLM, an accessory phosphoprotein, regulates the activity of both NCX1 and NKA when phosphorylated at Ser-68. PP1 has been shown to modulate pSer-68-PLM regulation of NKA, but it is unknown whether this mechanism is involved in NCX1 regulation. In this study we hypothesized that a direct and functional NCX1-PP1c interaction is a prerequisite for dephosphorylating the pool of pSer-68-PLM which interacts with NCX1. We aimed to identify the molecular mechanisms and functional consequences of the NCX1-PP1c-PLM interaction in the heart.

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Paper 3

Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1)

NCX1 has been shown to bind to pSer-68-PLM, which inhibits NCX1 activity. Other studies, however, have failed to demonstrate a functional interaction of these two proteins. In this study we hypothesized that NCX1 and PLM are direct, functional interacting partners. We aimed to design a high affinity blocking peptide targeted towards the pSer-68-PLM-NCX1 protein interaction, and to analyze the biological function of this interaction.

Paper 4

Mapping the in vitrointeractome of cardiac Na⁺-Ca²⁺exchanger 1 (NCX1)

Dysregulation of NCX1 is observed in human end-stage HF. Currently, no selective NCX1 modulators are in clinical use. The cytosolic loop of NCX1 mediates its regulation by associating with various cytosolic factors. In this study we hypothesized that elucidation of NCX1 protein interactions will broaden our understanding of its regulation and this knowledge may be of use in developing specific NCX1 modulators. We used a proteomic approach to screen for novel NCX1 interacting partners.

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3 Methodological considerations

This section discusses key aspects and considerations for the methods used in the presented papers.

Various molecular biology and biochemical techniques were employed on tissue from human patients and animal models of cardiac disease for in vivo experiments. We also used primary cell cultures and immortalized human cells for in vitroexperiments. Computational methods were used for functional annotation and construction of protein-protein interaction networks, while biophysical methods were used to elucidate important mechanisms.

3.1 Human myocardial biopsies

To quantify and demonstrate the clinical relevance of calpain dysregulation in paper 1, we used explanted human left ventricle (LV) biopsies in western blot analysis. The LV biopsies were from patients undergoing elective aortic valve replacement surgery for treatment of severe, symptomatic aortic stenosis (AS). In AS, calcification of aortic valves results in aortic valve narrowing, which constricts blood flow into the LV. This constriction increases afterload and ventricular wall stress, which stimulates hypertrophy of the LV. This hypertrophic remodeling initially restores wall stress, which preserves the LV function, however, over time, the heart is unable to maintain this high pressure gradient. Reduced systolic function ensues and progresses to HF (153). Biopsies from AS hearts therefore serve as a model for hypertrophy, suitable for studying how signaling is altered in progression to HF. This is advantageous compared to using tissue from patients undergoing heart transplants, as this usually represents end-stage HF. Obtaining control samples from healthy hearts is not feasible due to the invasiveness of the procedures and ethical considerations. Instead, as negative controls, we used biopsies from patients with coronary artery disease (CAD), undergoing coronary artery bypass graft operation (CABG), as this represent a different pathology from AS. Specifically, biopsies where taken from non-ischemic areas in CAD patients to ensure viable tissue that has not progressed to HF. A major limitation with the use of human samples is the low sample numbers.

Informed written consent was obtained from all the patients. The protocol conformed to the Declaration of Helsinki and was approved by the Regional Committee for Research Ethics in Eastern Norway (Project 2010/2226).

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3.2 Animal models

Tissue from rat LV was used in all the papers contained in this thesis. Specifically, in papers 1-4, molecular biology techniques were applied on wild type (WT) rat LV tissue to investigate normal physiology.

In paper 1-3, the aortic banding (AB) rat model (a model for AS) was used to study molecular changes that occur during the development of LV hypertrophy and progression to HF. As control we did not use WT animals, instead we used banded animals, where the silk suture around the ascending aorta was not tightened (sham-operated control animals). This controls for the effects related to the surgery (technical control).

There are many advantages of using rats in cardiovascular research: Firstly, its physiology in pathological conditions is similar to the corresponding human condition (154). Additionally, rat models offer the opportunity to study molecular changes in cardiomyocytes under relatively controlled experimental conditions compared to tissue obtained from human patients which may have comorbidities. From a practical perspective, rats are inexpensive and require little space compared to larger animals. They also have short life cycles and gestation periods which allow for the generation of large sample sizes in a short period of time (155). There is good reproducibility of cardiac hypertrophy with the use of rats, and a low mortality rate after the surgery, compared to for example mice, as rats have ~10 times larger hearts (156). The large hearts also provide more tissue for biochemical experiments. The use of rats is, however, not without limitations: One striking difference from humans is that the heart rate in humans is 60-90 bpm, compared to 250-493 bpm in rats (157). This means that the rats have a much shorter action potential than humans, indicating differences in the ion transport during the excitation-contraction coupling. Indeed, more cytosolic Ca²⁺removal is mediated by SERCA2 in rats compared to humans (17,155). Another drawback of using rat models is that they do not necessarily follow the usual disease progression as it happens in humans. The development of heart disease in humans is influenced by exposure to various risk factors throughout the individual’s life, but in animal models, disease states are induced over relatively short periods of time without the accompanying comorbidities. Also, humans are often taking a cocktail of medicines and are typically old, whereas young animals are used in experiments, hence the influence of medicines and age related factors may not be properly simulated by such models.

All animal experiments were approved by the Norwegian Animal Research Committee (FOTS ID 3820; 7714) and conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1996).

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3.3 In vitro cell models

In vitro mammalian cell models are the most commonly used source for protein research as they minimize the use of live animals. In addition, they allow for the manipulation of cellular physiology in order to elucidate molecular mechanisms. In the papers contained in this thesis we made use of three mammalian cell lines: Neonatal and adult rat cardiomyocytes were used as a source of primary culture, while human embryonic kidney 293 (HEK293) cells was the immortalized cell line used. Primary cultures are cells that are freshly isolated from organs and consist of a heterogeneous population of cells. Digestive enzyme (Collagenase type 2) is used to degrade the extracellular matrix of the heart. In neonatal rat hearts, due to their size the hearts are treated with the enzyme and the tissue is gently tweezed apart to yield the cells which are maintained in culture for up to five days, based on the seeding density we employ (3.75 x 10⁵ ml^-1). The neonatal cells were isolated from 1-3 day old rats and these cells were used to investigate the localization of the NCX1-calpain, NCX1-PP1c and NCX1- PLM interaction, using the fractionation kit. In addition, we also used these cells to investigate calpain cleavage after Ca²⁺stimulation. The main advantage of these cells is that they are easier to culture and provide a controlled environment for studying the effects of biochemical interventions. One drawback is that these cells are not terminally differentiated. This means that the contractile machinery is not developed. Therefore, we also made use of adult cardiomyocytes.

To obtain adult cardiomyocyte cells, the enzyme is perfused through aorta and coronary artery using Langendorff apparatus. Adult cardiomyocyte cells are more difficult to culture, therefore freshly isolated adult cardiomyocytes were used in experiments. These cells are differentiated, and subcellular structures are completely formed. Since cardiomyocyte morphology changes with time, cells were isolated and used in further assays on same day. Unlike neonatal cardiomyocytes, healthy isolated adult cardiomyocytes are rod shaped, striated, and do not exhibit spontaneous contractions. However, the isolation procedure is difficult, and cell quality and yield depend on the mounting of the heart on the Langendorff perfusion system. There is also variation in the quality of the digestive enzyme (collagenase) activity from batch to batch. To assess cell quality, we use visual inspection (cells must be rod shaped, have clear striations, have intact membranes, look viable and not have spontaneous contractions. One limitation is that results obtained from these cells cannot be extrapolated to tissue or organ level.

Immortalized cell lines (HEK293) were used in loss-of-function and gain-of-function experiments. A general advantage of using cell lines is that it allows for the study of cell responses in isolation, without neurohormonal, mechanical and paracrine effects. Transfection of plasmids with the Ca²⁺ method described in paper 2 is used to express a modified protein into the cells. Adult cardiomyocytes have a low transfection efficiency, therefore HEK293 cells were used. The method facilitated investigation of NCX1-specific effects, as HEK293 cells have been shown to be devoid of

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endogenous NCX1 (158). An advantage of using HEK293 cells is their high yield in protein expression (30-50%) (139), as adult cardiomyocytes have been shown to have a low transfection efficiency when plasmids are used. They also facilitate reproducibility and are easy to maintain in culture. HEK293 cells were also used in the functional analysis of NCX1 activity, using the patch clamp method.

3.4 Biochemical and molecular biology techniques

Several molecular biology techniques were used to elucidate the biological function of the novel NCX1 regulators.

3.4.1 Fractionation

In paper 1-3, we used subcellular fractionation to determine the distribution of NCX1 and its protein partners. The commercially available compartment protein extraction kit (2145, Merck Millipore Billerica, MA, USA) separates proteins into their various subcellular locations, based on their solubility in different buffer-and detergent conditions. Western blot was used to visualize the subcellular localization of the proteins. This method was employed early in our studies to determine if the novel partner was in the same subcellular space as NCX1, which is in the membrane.

3.4.2 Co-immunoprecipitation (Co-IP) and pull-down assays

Co-IP’s (paper 1-4) and pull-down assays (paper 1-3) are popular techniques for identifying protein- protein interactions. In Co-IP’s, specific antibodies are used to capture proteins that are bound to a specific target protein and the protein complexes are visualized by Western blot. The method identifies both direct and indirect protein binding partners (Fig. 6). One is more likely to achieve success with high abundant and stable interactions. Immunoprecipitation is an inexpensive method, however each interaction must be optimized in order to detect the bound proteins. Nonspecific binding is a problem often encountered, but can be reduced by changing the ionic strength and detergent conditions of the wash buffer used. One could also increase or decrease the amount of primary antibody used to capture the complex. False negative results can be attributed to too stringent washing conditions or the protein binding site overlaps with the antibody epitope. Antibody heavy and light chain contamination can also obscure the results, but this interference can be eliminated by using heavy and light chain specific secondary antibodies which unmask the heavy and light chain regions on polyvinylidene difluoride (PVDF) membranes. Further, one could covalently couple the antibody to the beads. In all our Co-IP experiments, species-specific non-relevant antibodies or blocking peptide (block antibody epitope) was used as negative control. Immunoprecipitation, as it is normally performed, does not provide quantitative data regarding the affinity or stoichiometry of an interaction, therefore other methods are required to determine this.

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Figure 6. Schematic illustration showing the principle of Co-IP. The IP antibody, specific for protein X, is immobilized on protein A/G beads. The latter is a purified A/G recombinant fusion protein that has been covalently immobilized onto agarose beads and enables the capture of antibodies from a wider range of species and isotypes. When the lysate is added to the immobilized antibody, protein X is captured and co-precipitates protein Y by directly binding to protein X (left figure) or binds indirectly through the bridge protein, protein Z (right figure). The protein complexes are visualized by Western blot.

A pull-down assay is an affinity purification method that is similar to immunoprecipitation and can also be used to detect direct protein/peptide interactions. This method was used to validate the interactions identified by the Co-IP and peptide array method. This method uses exogenous proteins tags such as biotin, FLAG, histidine (His) and glutathione S-transferase (GST) as bait (159). In a typical experiment, the monoclonal anti-biotin beads are incubated with biotinylated peptides and the recombinant protein of interest (Fig. 7). As negative control the recombinant protein is incubated with the monoclonal anti-biotin beads however the biotin peptide is not added. The protein complexes are visualized by Western blot

Figure 7. Schematic illustration showing the principle of a pull-down assay. Anti-biotin beads are co- incubated biotinylated peptides. After which the recombinant proteins is added. Detection of the interaction is visualized by Western blot.

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3.4.3 Peptides

In paper 1-4 we made use of peptide arrays to map antibody epitopes and also to map NCX1 partner interaction down to amino acid level. Soluble peptides were used to further validate binding by pull- down assays.

The protein of interest is synthesized as 20-mer overlapping peptides on cellulose membrane (Fig. 8). To investigate binding, the membrane is then incubated with recombinant protein partner, biotinylated peptides in solution or an antibody. Binding is visualized by Western blot analysis and is semi-quantitative.

Two dimensional peptide arrays, where the amino acids in the native peptide sequences were systemically replaced with every possible amino acid, were used to develop the high affinity NCX1- PLM disruptor peptide.

Several considerations must be kept in mind in the interpretation of peptide array data. 1) Signal intensity of the spot is affected by homogeneity of the peptide synthesis. 2) Excess amount of protein on the membrane might give non-specific binding. 3) Because the protein is synthesized as a linear sequence, this might create false binding sites as sites which are inaccessible in the folded protein become accessible.

Figure 8. Schematic representation of a peptide array synthesis. A linearized protein sequence of the protein of interest is synthesized as overlapping peptide fragments on a membrane. Each spot represents the overlapping peptide sequence of certain length e.g. 20 amino acids with a 3 amino acid offset.