Doctoral Dissertation

UNIVERSITY OF COPENH AGEN FACULTY OF HEALTH A ND MEDICAL SCIENCES

Kirstine Callø

The Transient Outward Potassium Current in Healthy and Diseased Hearts

December 2017

The Faculty of Health and Medical Sciences at the University of Copenhagen has accepted this dissertation, which consists of the already published dissertations listed below, for public defence for the doctoral degree in Veterinary Science. Copenhagen, 24th of October 2018. Professor Ulla Wewer, Dean

Public defence will be held in auditorium A1-05.01, Dyrlægevej 100, Frederiksberg Campus, University of Copenhagen, Friday 11th of January 2019 at 1 p.m.

Kirstine Callø Section for Anatomy, Biochemistry and Physiology Department of Veterinary and Animals Sciences Faculty of Health and Medical Sciences University of Copenhagen [email protected]

Copenhagen 30.12.2018 ISBN 978-87-971049-0-3

1 1 Contents

2 PUBLICATIONS...... 3 3 PREFACE ...... 4 3.1 Conflict of interests ...... 4 4 DANSK RESUMÉ...... 5 5 SUMMARY IN ENGLISH ...... 7 5.1 Abbreviations...... 9 5.2 Overview of cardiac currents and ion channel subunits ...... 10 5.3 Nomenclature ...... 11 5.3.1 Currents ...... 11 5.3.2 Ventricular layers ...... 11 6 INTRODUCTION - ELECTRICAL ACTIVITY OF THE HEART...... 12 6.1 Aim and hypotheses...... 12 6.2 The electrocardiogram, ECG ...... 12 6.3 The cardiac conduction system ...... 13 6.3.1 Purkinje networks ...... 14 6.4 The cardiac action potential and underlying ionic currents ...... 16 6.4.1 Nodal action potentials ...... 17 6.4.2 Atrial action potentials ...... 18 6.4.3 Action potentials in the His-Purkinje system ...... 19 6.4.4 Ventricular action potentials ...... 20 6.5 Excitation-contraction ...... 21 6.6 Neurohumoral regulation ...... 22 6.6.1 Sympathetic regulation of the heart ...... 23 6.6.2 Parasympathetic regulation of the heart ...... 24 7 REGIONAL HETEROGENEITY OF VENTRICULAR ACTION POTENTIAL MORPHOLOGY AND IMPLICATIONS FOR CARDIAC FUNCTION ...... 26 7.1 Transmural heterogeneity in early repolarization ...... 26 7.2 The role of the transient outward potassium current in excitation-contraction coupling ...... 28 7.3 The transient outward potassium current, Ito ...... 31 7.3.1 Molecular composition of Ito ...... 31 7.3.2 Pharmacology of Ito,f ...... 32 7.3.3 The effect of Ito activators NS5806 and NS3623 on isolated canine ventricular cells ...... 33 7.4 Pathophysiological implications of Ito,f ...... 37 7.4.1 Ito,f and cardiac arrhythmias ...... 37 7.4.2 Ito,f in heart failure ...... 39 7.5 Is pharmaceutically increased Ito,f potentially beneficial in failing hearts? ...... 42 8 CONCLUDING REMARKS AND FUTURE PERSPECTIVES ...... 45 8.1 The physiological significance of the Purkinje fibre distribution ...... 45 8.2 Is pharmaceutically increased Ito,f potentially beneficial in heart failure? ...... 46 8.3 Methological considerations ...... 47 8.4 Conclusion ...... 48 9 ACKNOWLEDGEMENTS ...... 48 10 REFERENCES ...... 49

2 2 PUBLICATIONS

The dissertation is based on the following publications:

A dual potassium channel activator improves repolarization reserve and normalizes ventricular action potentials. Calloe K, Di Diego JM, Hansen RS, Nagle S, Treat JA, Cordeiro JM. Biochem Pharmacol. 2016 May 15;108:36-46

Tissue-specific effects of acetylcholine in the canine heart. Calloe K, Goodrow R, Antzelevitch C, Olesen SP, Cordeiro JM. Am J Physiol Heart Circ Physiol. 2013 Jul 1;305(1):H66-75.

Physiological consequences of transient outward K+ current activation during heart failure in canine left ventricle. Cordeiro JM*, Calloe K*, Moise NS, Kornreich B, Giannandrea D, Diego JDM, Olesen SP, Antzelevitch C. J *Contributed equally. J Mol Cell Cardiol. 2012 Jun;52(6):1291-8.

Comparison of the effects of a transient outward potassium channel activator on currents recorded from atrial and ventricular cardiomyocytes. Calloe K, Nof E, Jespersen T, Chlus N, Di Diego JM, Olesen SP, Antzelevitch C, Cordeiro JM. J Cardiovasc Electrophysiol. 2011 Sep;22(9):1057-66.

Effect of the Ito activator NS5806 on cloned Kv4 channels depends on the accessory protein KChIP2. Lundby A, Jespersen T, Schmitt N, Grunnet M, Olesen SP, Cordeiro JM, Calloe K. British J of Pharmacol. 2010; 160(8):2028-44.

Differential effects of the transient outward K+ current activator NS5806 in the canine left ventricle. Calloe K, Soltysinska E, Jespersen T, Lundby A, Antzelevitch C, Olesen SP, Cordeiro JM. J Mol Cell Cardiol. 2010; 48:191-200.

A transient outward potassium current activator recapitulates the electrocardiographic manifestations of Brugada syndrome. Calloe K*, Cordeiro JM*, Di Diego JM, Hansen RS, Grunnet M, Olesen SP, Antzelevitch C. *Contributed equally. Cardiovasc Res. 2009; 81(4):686-94. Editorial comment: in Cardiovasc. Res. 2009, 81:635-636.

3 3 PREFACE

The present thesis is based on studies performed at the Department of Veterinary and Animal Sciences (IVH) and the Department of Biomedical Sciences (BMI) at the University of Copenhagen (UCPH) and the Masonic Medical Research Laboratory (MMRL), Utica, NY, USA from 2008 to 2016.

I wish to express my gratitude to Professor Dan A. Klærke for his enthusiasm and encouragement. His way of approaching and exploring new scientific ideas is very inspiring and constantly reminds me that science is fun. Thank you for being a true mentor. I would also like to thank all members of Section of Anatomy, Biochemistry and Physiology at the Department of Veterinary and Animal Sciences. It is such a privilege to have so great colleagues; I truly appreciate our scientific and teaching collaborations. I would also like to thank my long term friends and collaborators, Drs Morten Bækgaard Thomsen and Morten Schak Nielsen at the Department of Biomedical Sciences, UCPH and Rie Schultz Hansen at Zealand Pharma. It is always a pleasure to work on projects together or talk science.

The proposed hypotheses are the result of my long lasting collaboration with Dr. Jonathan M Cordeiro and the data acquired during my research visits to the MMRL are the backbone in the present thesis. Jon was the first to point my attention to the role of the transient outward potassium current in calcium handling across the ventricular wall, and has been a constant source of inspiration. I enjoy our many discussions about science. I would also like to thank Dr. José Di Diego for introducing me to multicellular cardiac preparations, including the wedge model and transmural ECGs. I am grateful for the support of Dr. Charles Antzelevitch and the staff members at the MMRL for welcoming me and making Utica my second home.

Finally, I would like to thank my family for filling my life with fun and happiness.

3.1 Conflict of interests None

4 4 DANSK RESUMÉ

Formålet med denne afhandling er at beskrive den hurtige transiente udadgående kaliumstrøms

(Ito,f) rolle i raske og syge hjerter med fokus på mennesket og store dyr. Hjertets aktionspotentialer dannes spontant i sinusknuden og udbredes gennem forkamrene til atrioventrikulærknuden, derfra videre gennem det His'ske bundt i septum til Purkinjefibrene i hjertekamrene og endelig til det arbejdende myokardium. Hos primater og rovdyr, som menneske og hund, findes Purkinjefibrene udelukkende endokardielt, og depolariseringsbølgen spredes gennem myokardiet til epikardiet. Dette resulterer i en forsinket aktivering af epikardiet på 20-30 ms sammenlignet med endokardiet. På trods af forskellen i aktiveringstid kontraherer de forskellige lag af ventrikelvæggen synkront. Der er markante regionale forskelle i aktionspotentialernes form i ventrikelvæggens lag i hunde- og menneskehjerter: I epi- og midmyokardiet har aktionspotentialerne en fremtrædende tidlig repolarisering, hvilket resulterer i en "spike-and- dome"-morfologi. Spike-and-dome-morfologien findes ikke i endokardielle aktionspotentialer.

Denne forskel i den tidlige repolarisering skyldes en transmural gradient af Ito,f med lav/ingen Ito,f- ekspression i endokardiet og høj ekspression i mid- og epikardiet. Det er tidligere foreslået, at denne 2+ transmurale Ito,f gradient er vigtig for synkronisering af Ca -frigivelse og kontraktion på tværs af ventrikelvæggen. Hos hov- og klovdyr, som hest og svin, trænger Purkinjefibre dybt ind i ventrikelvæggen, hvilket resulterer i simultan aktivering af alle ventrikulære lag. I disse arter udviser aktionspotentialerne ikke en spike-og-dome-morfologi, og der er kun små transmurale forskelle i aktionspotentialernes form. Disse observationer giver anledning til:

Hypotese 1: En koordineret sammentrækning af endo-, mid- og epikardiet i ventrikulærvæggen kan skyldes anatomiske tilpasninger, såsom dybt penetrerende Purkinjefibre (som hos gris og hest) eller regionale forskelle i ekspressionsniveau af Ito,f hvilket resulterer i en transmural gradient i aktionspotentialernes tidlige repolarisering (som hos menneske og hund).

Hos menneske og hund er mange hjertesygdomme forbundet med ændringer i Ito,f. Øget Ito,f kan resultere i Brugada-syndromet, mens hypertrofi og hjertesvigt er associeret med et fald i Ito,f og en reduceret tidlig repolarisering i midt- og epikardiet. Reduceret Ito,f antages at bidrage til nedsat calciumhåndtering og kontraktion på grund af følgende mekanismer: i) I den enkelte hjertecelle vil 2+ reduceret Ito,f og dermed tidlig repolarisering medføre nedsat Ca influx gennem de 2+ spændingsafhængige L-type Ca -kanaler (ICaL), hvilket resultere i mindre og dårligt synkroniseret Ca2+-frigivelse (Ca2+-transienten) fra det sarcoplasmatiske reticulum (SR). ii) I menneske- og hundehjerter resulterer reduceret Ito,f i en reduceret transmural gradient af den tidlige repolarisering. Da denne gradient er vigtig for synkroniseringen af Ca2+-transienterne og cellulær kontraktion på tværs af hjertevæggen, kan dette yderligere forværre den kontraktile dysfunktionen af svigtende hjerter. Vi har identificeret og testet to stoffer som aktiverer Ito,f. Dette arbejde har resulteret i:

Hypotese 2: Farmakologisk forøgelse af Ito,f under hjertesvigt har flere gavnlige effekter: i) I den

5 2+ enkelte hjertecelle vil forøget Ito,f øge ICaL hvorved Ca -transienterne normaliseres og kontraktiliteten forbedres. ii) Ved at øge Ito,f, genoprettes den transmurale gradient i tidlig repolarisering, hvilket medfører en forbedret koordination af Ca2+-transienter og kontraktion på tværs af hjertevæggen. Dette reducerer hjertets energiforbrug og forbedrer derved hjertets effektivitet i forhold til iltforbrug.

6 5 SUMMARY IN ENGLISH

The aim of this dissertation is to describe the role of the fast transient outward potassium current

(Ito,f) in healthy and diseased hearts with a focus on the human heart and hearts from large animals. The electrical impulses of the heart are spontaneously generated in the sinoatrial node (SAN) and spread through the atria to the atrioventricular node (AVN), into the septum through the bundle of His, the bundle branches and finally to the Purkinje fibers and the working myocardium. In primates and carnivores, like human and dog, the Purkinje fibres are exclusively found in the endocardium and the depolarization wave has to travel through the working myocardium resulting in a delay in epicardial activation of approximately 20-30 ms. Yet, despite this delay, the ventricular layers contract in unison. There are marked regional differences in the configuration of the action potentials in human and canine hearts. The epi- and midmyocardium exhibit a prominent early repolarization resulting in a spike-and-dome morphology of the action potentials that is absent from the endocardium. This gradient is due to a graded expression of Ito,f. It has been proposed that the gradient in early repolarization is important to synchronize Ca2+ release and contraction across the ventricular wall. In ungulates, like horse and pig, the Purkinje fibres penetrate the entire ventricular wall, resulting in a simultaneous activation of all ventricular layers. In these species action potentials do not exhibit a spike-and-dome morphology and only minor differences are observed in action potential shape across the ventricular wall. These observations gave rise to:

Hypothesis 1: A coordinated contraction of the endo-, mid-, and epicardial layers of the ventricular walls can be the result of anatomical adaptations, such as deep penetrating Purkinje fibres (e.g. porcine and equine hearts) or regional differences in expression of Ito,f resulting in a transmural gradient in the early repolarization of the ventricular action potentials (e.g. human and canine hearts).

In human and canine hearts changes in Ito,f expression have been linked to cardiac disease. Increased

Ito,f may result in the Brugada Syndrome (BrS) whereas decreased Ito resulting in a reduced early repolarization is found in hypertrophic and failing hearts. This is thought to contribute to the impaired calcium handling and contraction by the following mechanisms: i) Reduced Ito,f and early repolarization result in decreased calcium current (ICaL), which in turn results in smaller and less synchronized Ca2+ transients in the individual cardiomyocytes. ii) In human and canine hearts the loss of Ito,f results in a loss of the transmural gradient of early repolarization. As this gradient is important for the synchronization of Ca2+ transients and cellular shortening across the ventricular wall this may further aggravate the contractile dysfunction of the failing heart. We have recently identified two activators of Ito,f and we propose:

7 in improve transmural coordination of Ca2+ transients and contraction. This decreases the energy expenditure of the heart and improves cardiac efficacy.

8 5.1 Abbreviations AF Atrial fibrillation AP Action potential APD Action potential duration ATP Adenosine triphosphat ATR1 Angiotensin II receptor type 1 ATII Angiotensin II AVN Atrioventricular node BCL Basic cycle length BPM Beats per minute BrS Brugada syndrome CaMKII Ca2+/calmodulin-dependent protein kinase II cAMP Cyclic adenosine monophosphate CICR Ca2+ induced Ca2+ release Cx Connexin DAD Delayed after-depolarization DAG Diacylglycerol DNA Deoxyribonucleic acid EAD Early after-depolarization EC Exciation-contraction ECG Electrocardiogram Endo Endocardium Epi Epicardium ER Endoplasmic reticulum GHK Goldman-Hodgkin-Katz HF Heart failure IP3 Inositol-1,4,5-tri-phosphate Kir Inwardly rectifying potassium channel KV Voltage gated potassium channel LA Left atria LV Left ventricle Mid Midmyocardium M1 Muscarinic receptor type 1 M2 Muscarinic receptor type 2 NaV Voltage gated sodium channel NCX Sodium calcium exchanger NFAT Nuclear factor of activated T-cells PIP2 Phosphaditylinositol-4,5-biphosphate PKA Protein kinase A PKC Protein kinase C PLB Phospholamban PLC Phospholipase C RA Right atria RNA Ribonucleic acid RV Right ventricle RVOT Right ventricular outflow tract

9 RyR Ryanodine receptors SAN Sinoatrial node SCA Spinocerebellar ataxia SERCA SR Ca2+ ATPase SR Sarcoplasmic reticulum SUD Sudden unexpected death TASK TWIK-related acid-sensitive K channel TWIK Tandem of P domains in a weak inward rectifying K channel VT Ventricular tachycardia VF Ventricular fibrillation 4-AP 4 aminopyridine

5.2 Overview of cardiac currents and ion channel subunits

Current α- Gene Alternative names β-Subunits Localization subunits

INa NaV1.5 SCN5A Naβ1 A+V If HCN4 Hyperpolarization-activated, Possibly KCNEx SAN+AVN cyclic nucleotide-gated cation channel; Pacemaker current, Ih ICaL CaV1.2-3 CACNA1C L-type, dihydropyridine  All cardiac tissue

/1D receptor

ICaT CaV3.2- CACNA1H T-type Not established SAN+AVN 3.3 /1 IKur KV1.5 KCNA5 Ultra rapid delayed rectifier Kvβ1-2 A (V?)

Ito,f KV4.3 KCND3 Fast transient outward KChIP2 A+V (Epi + Mid) potassium DPP6 KCNEx Ito,s KV1.4 KCNA4 Slow transient outward Kvβ A+V potassium IKs KV7.1 KCNQ1 Slow delayed rectifier KCNE1 possibly In all cardiac KvLQT others tissue IKr Kv11.1 KCNH2 hERG KCNE2 All cardiac tissue Rapid delayed rectifier

IK1 Kir2.x KCNJ2 Inward rectifier - V

IK,Ach Kir3.1/ KCNJ3 G-protein-gated Inward - SAN+AVN+A Kir3.4 KCNJ5 rectifier, GIRK IK,ATP Kir6.x KCNJ8 Inward rectifier SUR2B All cardiac tissue

Cardiac currents and ion channel subunits: Several of the β-subunits are promiscuous and may interact with more than one α-subunit (McKinnon and Rosati, 2016). A = atrial tissue, V = ventricular tissue, SAN = sinoatrial node, AVN = atrioventricular node, P = Purkinje fibers. Based on International Union of Basic and Clinical Pharmacology (IUPHAR) nomenclature (Gutman et al., 2005, Catterall et al., 2005a, Kubo et al., 2005, Hofmann et al., 2005, Catterall et al., 2005b).

10 5.3 Nomenclature

5.3.1 Currents Often currents recorded from ion channel subunits expressed in heterologous systems do not fully recapitulate currents in native cells. In native cells other α- or β-subunits as well as other regulatory factors may modulate the current. To illustrate the complexity; the term Ito is used to describe a current recorded from ventricular cells. Based on voltage protocols or pharmacology Ito can be further subdivided into a fast component Ito,f and a slow component Ito,s. Ito,s is mediated by KV1.4 channels whereas Ito,f is mediated by KV4 subtypes (mainly KV4.2 and 4.3) plus different β-subunits including KChIP2. Currents mediated by heterologously expressed KV4.3 + KChIP2 is termed

IKv4.3+KChIP2 or KV4.3 + KChIP2 current.

5.3.2 Ventricular layers For canine wedge recordings (Di Diego et al., 2013) the endocardial layer (Endo) is defined as 0–3 mm from the endocardial surface and the epicardial layer (Epi) is defined as 0–3 mm from the epicardial surface of the tissue. The midmyocardium (Mid) is defined as the central 5 mm.

11 6 INTRODUCTION - ELECTRICAL ACTIVITY OF THE HEART

6.1 Aim and hypotheses The aim of the present dissertation is to describe the role of the fast transient outward potassium current (Ito,f) in healthy and diseased hearts. The Introduction includes a general description of the electrical activity in the hearts of large animals and a comparison of the electrical activation of ungulate hearts (exemplified by porcine and equine hearts) with hearts of primates and carnivores (exemplified by human and canine hearts, respectively). In the main part of the thesis the regional heterogeneity of ventricular action potential morphology and its implications for cardiac function are discussed with the main focus on Ito,f. Based on my findings and research from other groups the following hypotheses are proposed:

Hypothesis 1: A coordinated contraction of the endo-, mid-, and epicardial layers of the ventricular walls can be the result of anatomical adaptations, such as deep penetrating Purkinje fibres (e.g. porcine and equine) or regional differences in expression of Ito,f resulting in a transmural gradient in the early repolarization of the ventricular action potentials (e.g. human and canine hearts).

Hypothesis 2: Restoration of Ito,f in the setting of heart failure has several beneficial effects: i) 2+ 2+ Restoration of Ito,f increases Ca influx and Ca transients at the cellular level and thereby improve contractility. ii) By restoring Ito,f, the transmural gradient in early repolarization is restored resulting in improve transmural coordination of Ca2+ transients and contraction. This decreases the energy expenditure of the heart and improves cardiac efficacy.

6.2 The electrocardiogram, ECG The orderly pattern of depolarization and repolarization during the cardiac cycle gives rise to the characteristic deflections on the electrocardiogram (ECG). In its simplest version, the ECG can be obtained by placing electrodes on three limbs as in Einthoven's triangle, where lead I represents the voltage difference between the right and left arm (or foreleg), lead II the difference between the right arm and the left leg (or hind limb) and lead III the difference between the left arm and the left leg (Figure 1A). From these electrodes, the unipolar augmented limb leads aVR, aVL and aVF ("a" for augmented, "V" for vector, "R" for Right, "L" for left and "F" for foot) can be obtained. Together these leads form the hexaxial reference system (Figure 1B).

Figure 1: Electrical activity in the heart: (a) Einthoven's triangle showing the axis of the bipolar leads I-III. Modified from (By Npatchett). (b) The vector relationship of the limb and augmented unipolar leads. (c) Action potential waveforms in different areas of the heart. The latency approximates that of the healthy human heart. Reprinted from Cardiovacular Research (Hibino et al., 2010).

In addition, precordial leads (V1-V6) are often added, resulting in the 12 lead ECG. A depolarizing wave moving toward a positive electrode will result in a positive deflection on the ECG. The heart's electrical axis is the general direction of the ventricular depolarization wave front and can be determined by identifying the lead with the largest positive amplitude of its R wave. The ECG intervals in different leads may vary and often lead II is used as the standard lead. In lead II, the depolarization of the atria results in a positive deflection, the P wave and the QRS complex reflects the rapid depolarization of both ventricles1. The T wave represents ventricular repolarization and hence, the QT interval represents the period the ventricles are depolarized. Figure 1C shows the timing and configuration of action potentials in different regions of the heart and their reflection on the ECG. Thus, the ECG conveys a large amount of information about the electrical properties of the heart as well as its structure and position.

6.3 The cardiac conduction system The cardiac conduction system consists of a network of specialized myocardial cells that generates the cardiac rhythm and assures a fast and coordinated propagation of the electrical impulse resulting in an efficient contraction of the heart. The normal cardiac impulse is generated by spontaneous depolarization of specialized pacemaker cells in the sinoatrial node (SAN). The human SAN is a crescent-shaped structure located subepicardially at the junction of the right atrium and the superior vena cava and extending along the crista terminalis (James, 2002). The depolarizing impulse propagates through the atria and initiates atrial contraction. Atrial contraction occurs late in the ventricular diastole where the pressure in the ventricles is low, which allows opening of the

1 A Q wave is the initial negative deflection, an R wave is the initial positive deflection and the S wave is the first negative deflection after an R wave. A small letter denotes no or a small deflection, a large letter denotes a reflection of relative large amplitude.

13 atrioventricular valves. Normally atrial contraction confers a minor additive effect to ventricular filling. From the atria the depolarization reaches the atrioventricular node (AVN) located at the base of the atrial septum. The AVN serves several important functions: i) It provides a conduction delay between the atria and the ventricles. This allows the atrial systole to take place before the ventricular systole. ii) The AVN has a relatively long refractory period which protects the ventricles from atrial tachyarrhythmias and finally iii) the AVN can serve as a pacemaker due to intrinsic pacemaker activity; however, normally this activity is suppressed by impulses originating from the SAN. Distal to the AVN is the penetrating bundle which is embedded in the central fibrous body. The penetrating bundle emerges on the crest of the ventricular septum and becomes the bundle of His. The bundle of His bifurcates to form the right and left bundle branches. The bundle branches are electrically insulated from the underlying myocardium by connective tissue (James, 2002; Oosthoek et al., 1993b, 1993a). This ensures rapid conduction of the electrical impulses to the apex of the ventricles without activation of the base of the heart. The Purkinje network forms the terminal part of the cardiac conduction system. At specific sites the insulating sheaths are lost and the Purkinje network can depolarize the working myocardium.

6.3.1 Purkinje networks The Purkinje network can be divided into two components: The subendocardial fibres, which have connection to the bundle branches and assure the apex-to base activation of the ventricle and an intramural component consisting of deep penetrating fibres (Sedmera and Gourdie, 2014). These deep running Purkinje fibres penetrate the entire thickness of the ventricular walls and connections between the subendocardial and intramural network can be found at regular intervals (Oosthoek et al., 1993a). Intramural Purkinje fibres have been demonstrated in ungulates including sheep (Ryu et al., 2009), cow (Oosthoek et al., 1993a), pig (Sedmera and Gourdie, 2014), horse (Hamlin and Smith, 1965) and whale (Ono et al., 2009). In contrast, no intramural fibres have been found in dog, mouse or human hearts (James, 2003; Oosthoek et al., 1993a; Sedmera and Gourdie, 2014). The presence or absence of the intramural network does not appear to be related to heart size, as some small animals such as rats do have intramural fibres (Sedmera and Gourdie, 2014). The distribution the Purkinje network is physiologically important as the conduction velocity of electrical impulses is much higher in Purkinje fibres (2–3 m/s) than in myocardial cells (0.3–0.4 m/s) (Durrer et al., 1970; Pressler et al., 1982). Thus, the absence or presence of deep Purkinje fibres affects the activation pattern of the ventricular wall. Interestingly, Hamlin and Smith categorized domestic animals into two categories based on the activation pattern of ventricular depolarization (Figure 2).

14 Figure 2: Ventricular activation pattern of Category A and B hearts. Examples of Category A and B hearts, the arrows represent the direction and magnitude of the predominant QRS axis of the heart. Below are sagittal cross-sections of the heart viewed from the left lateral surface. The depolarized area is shown in black and arrows point the direction of the spreading. V10 and aVF are shown in their positions with respect to the heart and typical QRS complexes are shown. The thick line on the ECG marks the deflection corresponding to each drawing. (a) The three “states” of depolarization for species in Category A. The depolarization spreads through the endocardium from the apex to the free walls, and then from the endo- to the epicardium and finally the base and septum are depolarized (b) The two states of depolarization in species of Category B. The endocardium is activated first followed by a single burst of activation that excites the masses of the ventricles simultaneously. The second stage of ventricular depolarization proceeds simultaneously from many foci and in multiple directions and since no large dipoles are formed there is little contribution to the QRS complex of this second stage of activation. The depiction of ventricular activation pattern is from (Hamlin and Smith, 1965) with permission from John Wiley and Sons.

Category A includes primates and carnivores. They are characterized by a depolarization that spreads through the endocardium from the apex to the free walls, then from the endocardium to the epicardium and finally the base and septum are depolarized. Category B is represented by the ungulates, including cow, horse, pig, sheep and whales where the endocardium is activated first followed by a single burst of activation that excites the masses of the ventricles simultaneously. This burst of depolarization is caused by the deep penetrating Purkinje fibres (Hamlin, 2010; Hamlin and Smith, 1965). Based on the cytoachitecture of the Purkinje fibres and network, a separate Category C for rodents and lagomorphs has been suggested (Canale et al., 2012; Ono et al., 2009). In the following, the focus will be on large mammalian hearts, in particular the differences between Category A and B hearts. It should be noted that the ungulates do not represent a cladistic (evolution based) group but rather a phenetic group (similar, but not necessarily related) and some ungulates may be closer related to carnivores or primates than to other ungulates. See for example (Graphodatsky et al., 2011) for a depiction of the historic divergence relationships among the living orders of mammals.

The path of activation is reflected in the QRS complex on the body surface ECG (Figures 2 and 3). The deep penetrating Purkinje fibres allow the QRS complex of the porcine heart to be shorter than

15 that of canine hearts of equal size (Hamlin, 2007, 2010; Hamlin et al., 1975) as the transmural conduction velocity is faster in porcine hearts compared to canine hearts (Allison et al., 2007). Furthermore, the wave of depolarization producing the major body surface R wave potential is found in aVF in dog or V5 in man (lateral surface leads) suggesting that the depolarization wave propagates from the endo- to the epicardial surface in the left ventricular free-wall. In contrast, the free-walls of porcine hearts are activated almost simultaneously and the wave of depolarization producing the major body surface R wave potential is found in V10. Lead V10 is positioned on the seventh dorsal spinous process that registers potential difference in the apex to base direction (Hamlin, 2007). Other marked differences in the QRS complex between dogs and pigs can be found; in lead II the canine ECG has an qRs configuration whereas the porcine ECG exhibits a qrS configuration as shown in Figure 3 (Hamlin, 2010).

Though the activation pattern is similar in human and canine hearts there are differences in the ECG waveform that originate from the different orientation of the heart in the thoracic cavity, however, for leads facing comparable portions of the heart, the QRS complexes are similar in human and dog whereas the pig differs markedly (Hamlin and Smith, 1965).

Figure 3: Lead II ECG recordings from porcine and canine hearts: Reprinted from (Hamlin, 2010) with permission from Elsevier.

6.4 The cardiac action potential and underlying ionic currents The cardiac action potential is due to the orchestrated activation of different ionic currents. It can be divided into 5 phases: Phase 0, the depolarization phase due to activation of a rapid sodium current; Phase 1, the early repolarization phase due to activation of transient outward potassium currents; Phase 2, the plateau phase resulting from activation of calcium current as well the contribution of a persistent or late sodium current; Phase 3, the late repolarization phase due to the activation of delayed rectifying potassium current and inwardly rectifying potassium currents and finally; Phase 4, the resting phase where inwardly rectifying potassium currents and leaky potassium channels set the resting membrane potential close to the equilibrium potential for potassium. Not all phases are present in all cardiac cell types and there are marked variations between species; small animals such as mice have resting heart rates of 250-500 beats per minute (bpm), the action potentials are short and triangular without a clearly defined plateau phase resulting in an overlap of the early and the late repolarization phase. Large animals, like pigs, dogs and humans have slower

16 resting heart rates, 70-120 bpm (Merck Veterinary Manual), the action potentials are longer and have extended plateau phases. Action potential shape varies in different regions of the heart reflecting differential expression of ion channels and transporters (Figure 1C). Action potentials propagate in the heart via gap junctions. Gap junctions are comprised of connexins (Cx) that form cell-to-cell channels. This electrical coupling of the cells makes the heart work as a syncytium and will tend to even out differences in electrical potential between cells (Axelsen et al., 2013; Nielsen et al., 2012).

6.4.1 Nodal action potentials The action potentials of nodal cells (SAN and AVN) are markedly different from those in atrial or ventricular cells (Figure 4). The maximum diastolic potential is close to -60 mV and exhibits a spontaneous depolarization called the pacemaker potential which accounts for the intrinsic pacemaker activity. The pacemaker activity is at least partly due to activation of the "funny" current

(If) but cyclic release of calcium from the SR, the “calcium clock” also plays a role (Lakatta and

DiFrancesco, 2009). If is carried by hyperpolarization activated cyclic-nucleotide gated (HCN) channels that permit passage of both Na+ and K+ (Brown and Difrancesco, 1980). In human pacemaker cells, HCN4 is the predominant subtype (DiFrancesco, 2010). If depolarizes the membrane potential to the threshold for activation of voltage gated calcium channels. Because the depolarization is mediated by calcium currents, the action potential upstroke has a slow velocity and the amplitude is low. Both L- (Long lasting) and T- (Transient opening) type Ca2+ channels are present in the SAN. ICaL is responsible for the initiation of the action potential upstroke and is mainly carried by CaV1.3 channels. In contrast, ventricular ICaL is carried mainly by CaV1.2 channels. CaV1.3 activates at more negative potentials compared to CaV1.2 (Bartos et al., 2015) and this may be advantageous in pacemaker cells. T-type calcium channels (CaV3.1-3.3) contribute mainly to the late phase of the depolarization (Mesirca et al., 2015). The repolarization is due to inactivation of the calcium channels and activation of the delayed rectifier potassium currents IKr (Rapid) and IKs (Slow)

(Lei and Brown, 1996; Sanguinetti and Jurkiewicz, 1990), mediated by the KV11.1 (Sanguinetti et al.,

1995) and KV7.1 + KCNE1 channels, respectively (Barhanin et al., 1996; Sanguinetti et al., 1996). The acetylcholine activated inward rectifying current IK,Ach (mediated by Kir3.4 + Kir3.1) determines the excitability of the cells and is important for autonomic regulation of cardiac activity, see Section 6.6

(Calloe et al., 2007, 2013a). The inward rectifying potassium current IK1 mediated by Kir2 channels is absent from nodal tissue.

17 Figure 4: Action potentials from canine SAN cells: Spontaneous action potentials were recorded from isolated canine SAN cells. The recording is a courtesy of Dr. JM Cordeiro.

Due to the source-sink mismatch it is important that the SAN and AVN are electrically insulated from the surrounding polarized atrial myocardium. Differential expression of gap junction proteins is crucial for this insulation. The central nodes are devoid of the large and medium conductance connexins Cx40 and Cx43 that are responsible for efficient cell-cell coupling in the ventricles (Oosthoek et al., 1993b, 1993a), rather the small conductance Cx45 are expressed in nodal tissue (Honjo et al., 2002). This results in a relatively weak coupling of the nodal cells. Toward the periphery of the SAN the electrical coupling improves with expression of both Cx45 and Cx43 (Honjo et al., 2002).

6.4.2 Atrial action potentials Propagation of the depolarization from SAN cells to atrial cells through gap junctions results in the activation of large, rapidly activating sodium currents (INa) carried by NaV1.5 voltage gated sodium channels. This results in a rapid upstroke of the atrial action potential (Figure 5). NaV1.5 inactivates rapidly and the depolarization activates Ito,f carried by KV4.2/3 and Ito,s carried by KV1.4 channels

(Calloe et al., 2011) as well as the ultra-rapid current IKur mediated by KV1.5, resulting in an early depolarization. The rapid depolarization also activates ICaL carried by CaV1.2 channels that maintains the depolarization during the atrial plateau phase. Compared to ventricular cells, the atrial plateau potential is less depolarized. Atrial action potentials are often described as short and triangular in isolated cardiomyocytes, however, atrial action potentials recorded from intact tissue have a longer plateau phase and a duration comparable to ventricular action potentials (Calloe et al., 2011).

18 Figure 5: Action potential from canine right atria: Cells were isolated and paced at 1 s basic cycle length (BCL). Own recording. The experimental conditions are described in (Calloe et al., 2011, 2013b).

The late repolarization is a result of inactivation of CaV1.2 and a concomitant activation of voltage dependent potassium channels, including the delayed rectifying channels, IKr and IKs. The inward rectifier current IK1 contributes to the late phase of repolarization of the action potential and is important for setting the resting potential. IK1 expression is very low in the atria compared to the ventricles resulting in a resting membrane potential of approximately -80 mV compared to -90 mV in the ventricles (Calloe et al., 2013; Cordeiro et al., 2015; Schram et al., 2002). In contrast, IK,Ach is large in atrial cells where it determines the cellular excitability. IK,Ach is absent from ventricular cells (Calloe et al., 2007, 2013a). Other currents, including the small conductance potassium current mediated by SK channels (Diness et al., 2010), KV3 channels, the two-pore-domain potassium leak channels TWIK and TASK (Grandi et al., 2017) as well as different pumps also contribute to the membrane potential (Bartos et al., 2015). Due to differential expression of the repolarizing potassium channels, the action potential shape shows some heterogeneity in different areas of the atria (Burashnikov et al., 2004; Schram et al., 2002).

From the atria the depolarization wave reaches the AVN. The action potential configuration is reminiscent of action potentials in the SAN, however, the upstroke velocity is slightly faster (20 V/s), the resting membrane potential is a little more negative, approximately -65 mV, and the rate of the spontaneous depolarization is slower compared to SAN (Schram et al., 2002). The expression profile of ion channels is similar to the SAN, including expression of If, ICaL carried by CaV1.3 rather than

CaV1.2 and lack of INa and IK1. Rather than Cx43, the small conducting Cx45 gap junction isoform is expressed (Bartos et al., 2015). The expression of Cx45 and the absence of a functional INa result in a slow conduction velocity of approximately 5 cm/s through the AVN (Bartos et al., 2015). The conduction delay in the AVN permits the atrial systole to occur before the ventricular systole.

6.4.3 Action potentials in the His-Purkinje system After traversing the AV node the electrical impulses reach the bundle branches and the His-Purkinje system (Figure 6). The Purkinje fibres are optimized for rapid conduction (2 m/s) (Bartos et al., 2015),

19 which is reflected in the abundant expression of both the large- and intermediate-conductance gap junctions, Cx40 and Cx43.

Figure 6: Action potential from canine Purkinje fibre: Cells were isolated from free-running Purkinje strands and paced at 1 s BCL. Own recoding, the experimental conditions are described in (Calloe et al., 2013).

Another contributing factor to the fast conduction is large NaV1.5 currents resulting in a fast velocity of the AP upstroke (400-800 V/s) (Dun and Boyden, 2008; Schram et al., 2002). The resting membrane potential is very negative (approximately -90 mV), increasing the available INa. The upstroke is followed by a repolarization due to rapid inactivation of INa and activation of Ito (Dumaine and Cordeiro, 2007). The plateau phase is less depolarized compared to ventricular cells (Dumaine and Cordeiro, 2007), likely as a result of a lower ICaL expression. The early repolarization and plateau phases determine the amplitude of IKr. At fast pacing the early repolarization is reduced as there is less time available for Ito to recover from inactivation resulting in a more positive plateau phase.

This in turn results in a larger amplitude of IKr and a shortening of the action potential duration (APD)

(Dumaine and Cordeiro, 2007). At slow rates Ito is large and IKr is small resulting in longer action potentials. This suggests Ito play a role in rate adaptation of the APD. In general, Purkinje fibres have longer APD compared to ventricular cells (Calloe et al., 2013), possibly due to lower expression of

IKr, IKs, and IK1 (Atkinson et al., 2011). A spontaneous phase 4 depolarization is found in Purkinje fibres proximal to the bundle branches (Bailey et al., 1972), however, in free running Purkinje strands from canine ventricles the resting membrane potential is stable (Calloe et al., 2013).

6.4.4 Ventricular action potentials Compared to the atria the resting membrane potential of ventricular cells is more negative, approximately -90 mV (Figure 7). The action potentials have a fast upstroke velocity (∼350 V/s) due a high expression of NaV1.5 channels (Calloe et al., 2011; Schram et al., 2002). Rapid inactivation of

NaV1.5 and concomitant activation of Ito,f mainly carried by KV4.3 with the auxiliary β-subunit KChIP2 underlies the early repolarization resulting in a spike-and-dome morphology of action potentials in mid- and epicardial layers (Calloe et al., 2008, 2010, 2011a). In large animals, Ito contributes little to the plateau phase and late repolarization due to its rapid inactivation kinetics. However, in smaller

20 animals, such as mice and rats, with short action potentials Ito constitutes a major late repolarizing current (Grubb et al., 2014). The plateau phase is due to activation of the L-type channel CaV1.2.

Figure 7: Action potentials from canine left ventricular Endo and Epi cells: Cells were isolated from LV and paced at 1 s BCL. The currents underlying the ventricular action potential are shown on the Endo AP. Endo has a longer APD compared to Epi. The depicted Epi action potential exhibits the classical "Spike-and- dome" morphology and the magnitude of the early repolarization (the "notch") depends on Ito expression. Own recordings, the experimental conditions are described in (Calloe et al., 2011, 2013a). The repolarization is a result of inactivation of CaV1.2 and the activation of IKr and IKs and finally IK1 (Bartos et al., 2015). The plateau phase is assisted by special properties of these repolarizing currents; IKs activates slowly, IKr activates rapidly, but the activation is overlapped with a rapid inactivation process. This inactivation is first released when the membrane potential starts to repolarize at the end of the plateau phase allowing IKr to contribute the late repolarization. IK1 is strongly inwardly rectifying resulting in little current during the plateau phase (Calloe et al., 2013;

Cordeiro et al., 2015; Grandi et al., 2017). Besides contributing to the late repolarization, IK1 clamps the resting membrane potential close to the equilibrium potential for potassium. In many species the action potential configuration differs markedly in different regions of the ventricles (Figure 7). This will be discussed in details in Section 7.

6.5 Excitation-contraction In the ventricle the majority of the L-type Ca2+ channels are located in the T-tubules facing clusters of ryanodine receptors (RyR) in the SR (Figure 8). These clusters may contain more than 100 RyRs. The influx of Ca2+ ions through L-type Ca2+ channels during the plateau phase opens these RyRs and triggers a much larger Ca2+ release from the SR, this process has been coined the Ca2+ induced Ca2+ release (CICR) (Bers, 2002). In humans, approximately 30 % of the increase in intracellular Ca2+ is due to influx through L-type Ca2+ channels. The combination of Ca2+ released from the SR and Ca2+ entering the cell raise the free intracellular Ca2+ concentration ten times; from a diastolic level of 100 nM to a peak systolic level of 1 μM (Bers, 2002). The release of Ca2+ from a single RyR is believed to be the elementary event underlying excitation-contraction (EC) coupling in cardiac muscle (Cheng et al., 1996). Activation of 6-20 RyRs in a cluster results in a Ca2+ spark (Bridge et al., 1999). The Ca2+ transient represents the spatial and temporal summation of many Ca2+ sparks (Cannell et al., 1995; Santana et al., 1996). During the Ca2+ transient, cytosolic Ca2+ binds to troponin C, exposing the binding site for myosin on the actin filaments, the sarcomeres shortens and the cells contract.

Figure 8: Excitation-contraction (EC) coupling and Ca2+ induced Ca2+ release (CICR). Ca2+ transport in a ventricular cardiomyocyte, see text for details. The insert shows the time course of an action 2+ 2+ potential, the Ca transient [Ca ]i and contraction in a rabbit ventricular myocyte. The amount of Ca2+ buffered in the cytosol is not shown. Reprinted from (Bers, 2002) with permission from Nature Publishing Group.

Besides activation of L-type Ca2+ channels, other sources of Ca2+ can trigger CICR. Activation of T-type Ca2+ current or sodium calcium exchanger (NCX) operating in "reverse-mode" are also capable of initiating SR Ca2+ release and cellular contraction (Sipido, 1998), however, the physiological relevance of these alternative triggers of the CICR has been questioned (Bers, 2002). For relaxation to occur Ca2+ has to be transported out of the cytosol, either by being sequestered into the SR by the SR Ca2+-ATPase (SERCA) or extruded from the cell via the NCX and the Ca2+-ATPase in the plasma membrane or finally Ca2+ can be removed by the mitochondrial Ca2+ uniporter (Bers, 2002). As the NCX exchanges three Na+ for one Ca2+ it results in a depolarizing current that contributes to the plateau phase, whereas the Ca2+-ATPase and the sodium potassium ATPase (Na+/K+-ATPase, moves three Na+ out for every two K+ in) contribute with repolarizing currents (Bers, 2002). Cardiac contractility can be modulated: i) By altering the amplitude or duration of the Ca2+ transient or ii) By changing the Ca2+ sensitivity of the myofilaments. The Ca2+ sensitivity of the myofilaments is dynamically changed by stretching of the cardiac tissue. This is important for the heart to adapt to increased diastolic filling and underlies the classical Frank-Starling response (Bers, 2002). The amplitude and duration of the Ca2+ transient can be modified by action potential shape as discussed in Section 7.2. Neurohumoral stimulation can modulate both the Ca2+ transient morphology as well as the Ca2+ sensitivity of the myofilaments.

6.6 Neurohumoral regulation The heart is under constant autonomic regulation and the activity of many ion channels is modulated by catecholamines and acetylcholine. Additionally, many voltage gated ion channels exhibit a strong time-dependence due to activation or deactivation kinetics or a time-dependent release from inactivation. This implies that changes in the heart rate will also directly affect many currents involved in the cardiac action potential.

22 6.6.1 Sympathetic regulation of the heart In humans activation of the sympathetic nervous system can increase the heart rate from approximately 70 to almost 200 bpm. The effect of the sympathetic system is mediated by catecholamines released from postganglionic neurones (noradrenaline) or from the chromaffin cells of the adrenal medulla (adrenalin and noradrenalin) directly to the bloodstream. The β1 adrenergic receptor is the major effector in the heart, but other adrenergic receptors are also of importance.

Activation of β1 receptors triggers an intracellular cascade resulting in formation of cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA, Figure 9) and phosphorylation of multiple targets (Lundby et al., 2013) resulting in increased chronotropy, dromotropy, inotropy and lusitropy of the heart.

Figure 9: Autonomic regulation and cellular signalling. Binding of catecholamines (Adr) to β1 adrenergic receptors activate a stimulatory trimeric G protein leading to dissociation of the αs subunit. αs activates adenylate cyclases (AC), resulting in formation of cAMP from ATP. cAMP binds directly to HCN channels as well as other channels but most of its actions are due to activation of protein kinase A (PKA). PKA phosphorylates a whole range of proteins including many ion channels and components of the contractile machinery. The muscarinic receptor type 2 (M2) is the main mediator of parasympathetic stimuli. Acetylcholine (ACh) binding to M2 results in dissociation of αi from an inhibitory G protein and a decrease in AC activity. The βγ subunit can activate some isoforms of phospholipase C (PLC) and Kir3.1/3.4 channels. Activation of the M1 or the α1 adrenergic receptors results in dissociation of αq/11 subunit and activation of PLC. PLC hydrolyses PIP2 in the membrane resulting in the formation of inositol-tri-phosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC). IP3 binds to IP3 receptors in the SR, resulting in Ca2+ release. PIP2 is important for the function of many ion channels and transporters and the PLC mediated hydrolysis may change the activity of many proteins. This overview is simplified, not all receptors are present in all cells and other receptors and pathways are also of importance. Modified from the PhD thesis, Potassium Channels of the Heart, Calloe 2006.

The chronotropy of the heart is increased by binding of cAMP directly to the HCN channels resulting in increased If and an acceleration of the spontaneous depolarization in the SAN pacemaker cells (DiFrancesco, 1995, 2010). It is crucial to ensure adequate diastolic filling time during fast heart rates

23 and action potentials are markedly shortened by sympathetic stimulation; this is mainly due to PKA mediated phosphorylation of KV7.1 resulting in increased IKs (Chiamvimonvat et al., 2017; Marx et al., 2002; Volders et al., 2003). IKr is relatively insensitive to β1 stimulation (Thomas et al., 2004) but the effect of increased rate alone increases IKr (Rocchetti et al., 2001). Ito is reduced by adrenergic receptor activation (van der Heyden et al., 2006; Niwa and Nerbonne, 2010) and in addition, faster heart rates are associated with a reduced Ito due to insufficient time for recovery from inactivation (Calloe et al., 2008). This results in a decreased early repolarization, which is even further decreased by PKA mediated activation of CaV1.2 currents. The molecular mechanism underlying PKA mediated activation of CaV1.2 is not fully resolved (Weiss et al., 2013), but the increased ICaL enhance the CICR 2+ and the Ca transient amplitude. The increased ICaL results in increased dromotropy due to the increased conduction speed in the AVN. Other proteins involved in Ca2+ homeostasis are also phosphorylated, including troponin I, resulting in increased Ca2+ sensitivity and inotropy. The lysitropic effects are due to phosphorylation of troponin I and phospholamban (PLB). Phosphorylation of troponin I speeds up dissociation of Ca2+ from the myofilaments. PLB is an endogenous inhibitor of SERCA and phosphorylation of PLB relieves this inhibition, allowing faster removal of cytosolic Ca2+ and enhanced Ca2+ loading of the SR, which further increases the Ca2+ release and the amplitude of the Ca2+ transients. The faster relaxation combined with the increased Ca2+ levels facilitates a faster and more powerful contraction (Bers, 2002, 2008). However, the increased contractility comes at a price, cardiac efficacy is decreased as the oxygen consumption goes up (Bers, 2002).

6.6.2 Parasympathetic regulation of the heart The vagus nerve innervates the SAN and AVN as well as both atria. Cholinergic innervation of the ventricles is believed to be absent or sparse (Löffelholz and Pappano, 1985), though this view has been challenged in porcine hearts (Ulphani et al., 2010). Acetylcholine released during vagal stimulation has a marked negative chronotropic effect on the heart. This effect is principally mediated by acetylcholine receptors of the muscarinic type 2 (M2). The M2 receptors are predominantly expressed in atrial and nodal tissue and are sparse in the ventricles (Dhein et al.,

2001). They are generally coupled to pertussis toxin sensitive G proteins (G0/Gi). Binding of acetylcholine causes dissociation of the coupled G protein (Figure 9) and the αi/0 subunit inhibits the adenylate cyclase and reduces cAMP formation resulting in decreased If and automaticity. The reduction of cAMP decreases PKA activity, which in turn reduces Ca2+ currents and thereby slows

AVN conduction. The βγ subunits of the G0/Gi protein bind directly to Kir3.1 resulting in activation of IK,Ach (Krapivinsky et al., 1995b, 1995a; Logothetis et al., 1987). IK,Ach opposes the pacemaker current If and thereby reduces automaticity and slows the heart rate. The βγ-subunits also activate phospholipase C (PLC) causing hydrolysis of phosphaditylinositol-4,5-biphosphate (PIP2) into inositol-1,4,5-tri-phosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC), resulting in phosphorylation of a number of proteins, including ion channels. IP3 dissociates from the plasma membrane and binds to IP3 receptors in the ER resulting in Ca2+ release. PIP2 regulates the activity of many ion channels, including Kir3 and Kv7.1, thus, PLC mediated hydrolysis may affect the shape of the cardiac action potential (Hille et al., 2015).

25 7 REGIONAL HETEROGENEITY OF VENTRICULAR ACTION POTENTIAL MORPHOLOGY AND IMPLICATIONS FOR CARDIAC FUNCTION

7.1 Transmural heterogeneity in early repolarization Beside the noticeable differences between nodal, atrial and ventricular action potentials there are variations in action potential shape between the ventricles. Action potentials from the canine right ventricle have longer duration and a larger early repolarization compared to those from the left ventricle (Calloe et al., 2008; Di Diego et al., 1996) and differences in apex to base have been reported, with shorter APD in the apical regions in human and canine hearts (Chiamvimonvat et al., 2017; Janse et al., 2012; Ramanathan et al., 2006; Szentadrassy et al., 2005).

Figure 10: Transmural gradient of early repolarization in canine left ventricle. (a) Action potentials from the endo-, mid- and epicardium as well as a transmural electrocardiogram recorded simultaneously from a canine left ventricular wedge preparation at 1000 ms BCL. The early repolarization is minor in Endo action potentials whereas Mid and Epi action potentials exhibit a spike-and-dome morphology. Based on data from (Calloe et al., 2008). A drawing of the wedge preparation is depicted in the insert. (b) Representative Ito recordings from Endo, Mid and Epi cells isolated from canine left ventricle. Notice the difference in scale. The voltage clamp protocol is shown below. (c) Current-voltage relationship for Ito in the three different layers. Based on data from (Calloe et al., 2010).

In human (Drouin et al., 1995; Franz et al., 1987; McKinnon and Rosati, 2016; Näbauer et al., 1996; Szabó et al., 2005) and canine hearts (Calloe et al., 2008, 2010; Higuchi and Nakaya, 1984; Patel et al., 2009; Yan and Antzelevitch, 1996) there are marked differences in action potential morphology

26 across the ventricular wall (Figure 10 and Table 1). Action potentials from the mid- and epicardial layers have a pronounced early repolarization resulting in a "spike-and-dome morphology" that is lacking in the endocardium. The magnitude of the early repolarization mirrors the expression of Ito, with a high expression of Ito in epi- and midmyocardial layers. The transmural difference in early repolarization is reflected on the transmural ECG as a J-wave (Figure 10) (Calloe et al., 2008; Yan and Antzelevitch, 1996).

Table 1

Species Early repolarization Ito,f or KV4 gradient References heterogeneity Category A Human Medium Yes (Näbauer et al., 1996; Wettwer et al., 1994) Dog High Yes (Calloe et al., 2010; Pacioretty and Gilmour, 1995; Rosati et al., 2003) Cat Yes Yes (Furukawa et al., 1990; Harris et al., 2005; Schackow et al., 1995) Ferret Yes Yes (Brahmajothi et al., 1999; Dobrzynski et al., 2002; Patel and Campbell, 2005) Bear* Unknown Unknown (Gandolf et al., 2010) Category B Pig Absent Minor/Absent (Lacroix et al., 2002; Li et al., 2003; Rodríguez- Sinovas et al., 1997; Schultz et al., 2007; Stankovicova et al., 2000) Horse Absent** Minor/Absent (Finley et al., 2002, 2003; Pedersen et al., 2015) Cow Minor/Absent** Unknown (Carmeliet and Vereecke, 1969; Guo et al., 2008) Sheep Minor/Absent Unknown (Delmar et al., 1987)

Guinea pig Absent Absent (Guo et al., 2008; Varró et al., 1993; Zicha et al., 2003) Whale* Unknown Unknown (Meijler et al., 1992) Category C Rat . Ito,s and Ito,f contributes (Clark et al., 1993; Wickenden et al., 1999) to late repolarization Rabbit - Mainly Ito,s (Cheng et al., 2017; Fedida and Giles, 1991; Guo et al., 2008; McIntosh et al., 2000; Varró et al., 1993) Mice - Ito,s and Ito,f contributes (Grubb et al., 2014; Guo et al., 1999; Rossow et to latel repolarization al., 2009; Teutsch et al., 2007) *Categorized based on ECG morphology. **Action potentials recorded from epi- and/or midmyocardial regions do not have a marked phase 1 repolarization, suggesting that there is no early gradient in these hearts. Category C is included for comparison.

Cardiac action potentials from other animals, such as pig (Stankovicova et al., 2000; Rodríguez- Sinovas et al., 1997), cow (Guo et al., 2008) and horse (Finley et al., 2003; Pedersen et al., 2015) do not exhibit the spike-and-dome morphology and there appear to be minor/no differences in action potential shape from endo-, mid- and epicardial cells (Table 1 and Figure 11).

27 Figure 11: Action potentials from endo-, mid- and epicardium of porcine left ventricle. Action potentials were recorded from various regions at BCLs of 1, 2 and 5 s from porcine LV wedge preparations. Reprinted from (Rodríguez-Sinovas et al., 1997) with permission from Oxford University Press.

7.2 The role of the transient outward potassium current in excitation-contraction coupling In human and canine hearts (Category A based on Hamlin and Smith, 1965) the Purkinje network predominantly resides in the endocardium. The depolarization wave must then traverse the ventricular wall to excite the mid- and epicardial layers (Figure 12). Conduction in the working myocardium is relatively slow (0.3–0.4 m/s) resulting in a transmural conduction time of 20-30 ms in the canine left ventricle (Boukens et al., 2017; Calloe et al., 2008). In spite of this time difference in activation, all layers of the ventricle contract simultaneously. Interestingly, it has been found that the amplitude and kinetics of the ICaL in all three layers are similar when currents are activated by a standard voltage step protocol (Bányász et al., 2003; Cordeiro et al., 2004; Li et al., 2002). This indicates that ICaL as such is not important for the timing of the transmural contraction. Evidence suggests that the gradient of early repolarization is important for transmural coordination of 2+ contraction; cell contraction is initiated by influx of Ca through L-type CaV1.2 channels and subsequent CICR from the SR. Since CaV1.2 channels are voltage-gated, the shape of the action potential affects both the open probability (PO) of the channels as well as the electrochemical driving force for Ca2+ movement. This has elegantly been demonstrated using action potential clamp (AP clamp), a technique where pre-recorded action potentials are used to activate currents. AP clamp recordings of Ca2+ currents in isolated cardiomyocytes show that ICaL activated by action potentials with a prominent early repolarization, such as mid- and epicardial action potentials, has a larger peak and a greater total Ca2+ influx compared to ICaL activated by action potentials with little or no early repolarization, such as endocardial action potentials as shown in Figure 12 (Cordeiro et al., 2004; Harris et al., 2005; Sah et al., 2002, 2003).

Figure 12: Effect of the early repolarization on L-type Ca2+ current. L-type Ca2+ current was defined as the nicardipine- sensitive difference current. (a) Currents in response to a canine Endo (left) or Epi (right) action potential waveform were recorded from the same isolated canine LV Mid cell. To eliminate the effects of INa, the action potentials were modified so that the waveform started at –50 mV (top). Application of the Epi waveform produced a larger peak current and greater charge entry compared with the Endo waveform. (b) Mean data showing the response of ICa,L peak and integrated ICaL to the different action potential waveforms. Reprinted from (Cordeiro et al., 2004).

The action potential configuration will not only affect L-type Ca2+ currents but in turn also the shape and timing of the intracellular Ca2+ transients and cell shortening (Figure 13). The phase 0 depolarizing to +40 mV results in an almost maximal activation of CaV1.2 and the presence of a rapid early repolarization increases the electrochemical driving force for Ca2+ influx (Cooper et al., 2010; Cordeiro et al., 2004).

Figure 13: Effect of the early repolarization on Ca2+ transients and unloaded cell shortening. (a) Epi- or endocardial action potential waveforms were applied to isolated rat ventricular myocytes. Representative ICaL recordings are shown and 600 ms surface plots generated from confocal line scans. The line scans demonstrate the loss of recruitment and temporal synchronization of Ca2+ release events in myocytes stimulated with an Endo action potential waveform compared to an Epi waveform. Reprinted from (Sah et al., 2003) with permission from John Wiley and Sons. (b) Superimposed Endo and Epi action potential waveforms and corresponding cell shortening (bottom) recorded from a canine midmyocardial cell in response to the action potential waveforms (top). Five prepulses were applied to maintain a uniform SR Ca2+ content. The tick marks on the cell shortening traces denote the peak. The time to peak was 16.4±7.9 ms shorter using an Epi waveform. Reprinted from (Cordeiro et al., 2004).

2+ 2+ The relationship between local [Ca ]i and the probability of triggering a Ca spark (PSpark) is not linear (Cooper et al., 2010; Santana et al., 1996) and the increased Ca2+ influx associated with an early depolarization results in marked increase in PSpark (Cooper et al., 2010; Santana et al., 1996).

The increase in PSpark results in faster and more synchronized transients as shown in Figure 13A (Sah et al., 2003). The effect of the early repolarization on Ca2+ transients is also reflected on cell shortening. Figure 13B shows unloaded cell shortening of the same cell in response to AP clamp with either an endo- or epicardial waveform. Application of the epicardial waveform causes a faster time to peak and greater cell shortening (Cordeiro et al., 2004).

There are several implications of this: i) The early repolarization of the ventricular action potential 2+ 2+ determines the PO of the L-type Ca channels as well as the electrochemical driving force for Ca influx. ii) Ventricular cardiomyocytes with a large Ito resulting in a marked spike and dome 2+ morphology have a significantly larger peak ICaL as well as greater total Ca influx compared to cell types where Ito is absent iii) Variations in Ito across the ventricular wall contribute to differences in electrical and mechanical characteristics of the three cell types. The epicardium is the last region of the heart to be excited yet exhibits the fastest Ca2+ transient and cell shortening kinetics. As the activation sequence of the ventricular wall is from the endo- to the epicardium, the Ito gradient may synchronize transmural contraction by off-setting the conduction delay of 20-30 ms in animals with exclusively endocardial Purkinje fibres (Category A) as shown in canine hearts (Cordeiro et al., 2004). These results are at least partly supported by experiments on human ventricular wedges where action potentials and Ca2+ transients were optically mapped. In healthy hearts there was a tendency for a longer delay from action potential upstroke to Ca2+ transients in the endocardium compared

30 to in the mid- and epicardium (Lou et al., 2011). It should, however, be kept in mind that intrinsic differences in Ca2+ handling in the different layers exists, in particular, the decay of the Ca2+ transient are slower in endocardium (Cordeiro et al., 2007). Based on these observations Hypothesis 1 is proposed:

B hearts, e.g. porcine and equine hearts) or regional differences in expression of Ito,f resulting in a transmural gradient in the early repolarization of the ventricular action potentials (Category A, e.g. human or canine hearts).

7.3 The transient outward potassium current, Ito

7.3.1 Molecular composition of Ito In human and canine hearts the transmural gradient in early repolarization mirrors the amplitude of Ito, with large Ito in the mid- and epicardium (Calloe et al., 2010). Based on recovery time from inactivation, Ito can be divided into two separate currents: Ito,f (fast) and Ito,s (slow) (Brouillette et al.,

2004; Grubb et al., 2014; Näbauer et al., 1996; Shimoni et al., 1995). Ito,f predominates in the mid- and epicardium, whereas the slowly recovering Ito,s dominates in the endocardium (Akar et al., 2004;

Calloe et al., 2010; Näbauer et al., 1996). Ito,s has been ascribed to KV1.4, but KV1.5 may also contribute as KV1.5 mRNA has consistently been found in human and canine ventricles (Calloe et al.,

2010; Rosati et al., 2001; Zicha et al., 2004) and in many species the molecular identity of Ito,s remains poorly defined (McKinnon and Rosati, 2016). Ito,f channels consist of a tetramer of pore-forming KV4

α subunits (Figure 14) (Doyle et al., 1998). In human and canine hearts KV4.3 is the predominant α subunit of Ito,f (Akar et al., 2004; Calloe et al., 2010), but KV4.2 mRNA has been reported in human ventricle (Gaborit et al., 2007). Recently, a mutation in the gene encoding KV4.2 have been reported in a patient with a J-wave syndrome further supporting a functional role for KV4.2 in the human ventricle (Perrin et al., 2014).

Currents recorded from heterologously expressed KV4 channels do not recapitulate native cardiac

Ito,f and KV4 subunits have been shown to assemble with several β subunits and other regulatory factors (Lundby et al., 2010; McKinnon and Rosati, 2016; Niwa and Nerbonne, 2010). The K+ Channel

Interaction Protein 2 (KChIP2) increases KV4.3 current density by facilitating trafficking, slowing inactivation and accelerating recovery kinetics (An et al., 2000; Bahring et al., 2001; Foeger et al., 2013; Lundby et al., 2010). KChIP2 mRNA is more abundant in mid- and epicardium than in endocardium and this differential expression has been suggested to underlie the gradient in Ito,f (Calloe et al., 2010; Rosati et al., 2001, 2003). Besides KChIP2, the dipeptidyl-peptidases (DPP6 and

DPP10) interact with KV4 channels (Li et al., 2006; Nadal et al., 2003). DPP6 is expressed in the heart and similar to KChIP2, DPP6 enhances KV4.3 trafficking and accelerates recovery from inactivation.

In contrast to KChIP2, DPP6 accelerates KV4.3 inactivation (Lundby et al., 2010; Nadal et al., 2003) and increases single channel conductance (Kaulin et al., 2009). DPP10 mRNA has been detected in

31 atrial tissue (Turnow et al., 2015), but it is still unclear whether DDP10 is found in ventricular tissue (Cotella et al., 2010; Radicke et al., 2005).

Figure 14: Ito,f channel. The Ito,f channel consists of four KV4 α subunits. Each α subunit has six transmembrane segments. KChIP2 is thought to interact with the intracellular N terminus and may be involved in NS5806 binding. Besides KChIP2, DPP and KCNE subunits have been demonstrated to regulate channel function; however, the molecular background for 2+ these interactions is uncertain. CaMKII is activated by Ca /calmodulin and binds to the C terminus. Reported Ito,f loss- of-function mutations associated with spinocerebellar ataxia are shown in blue; gain-of-function mutations associated with the Brugada syndrome or atrial fibrillation are shown in red. All gain-of-function mutations are located to the C terminus that may be involved in stabilizing the interaction with KChIP2 (Callsen et al., 2005) and harbours several sites involved in post-translational modifications. Modified from (Cordeiro et al., 2016).

Several members of the promiscuous KCNE β subunit family modulates KV4 currents in heterologous systems and mutations in KCNE subunits affecting KV4.3 current density and/or kinetics have been implicated in cardiac arrhythmias in humans (Delpón et al., 2008; Levy et al., 2010; Lundby and Olesen, 2006; Nakajima et al., 2012; Ohno et al., 2011; Roepke et al., 2008; Wu et al., 2010). KCNE2 has been reported to induce an "overshoot" in KV4.3 peak currents during recovery from inactivation; the overshoot implying that the peak amplitude of the recovered current is transiently larger than that of the current activated by the reference pulse (Radicke et al., 2006). This overshoot was also reported for human epicardial Ito by the same group (Wettwer et al., 1994). However, for

KV4.3 and KCNE2 expressed in Xenopus laevis oocytes we did not observe this phenomena (Lundby et al., 2010) and Ito in canine cardiomyocytes does not exhibit an overshoot during recovery (Calloe et al., 2010). The reason for this discrepancy has not been resolved. Other subunits have been proposed to modulate Ito,f including the neuronal calcium sensor-1 (NCS-1) (Guo et al., 2002), KVβ

(Aimond et al., 2005), NaVβ (Deschênes et al., 2008; Marionneau et al., 2012) but the physiological importance of these interactions is still uncertain.

7.3.2 Pharmacology of Ito,f Several spider toxins containing three disulfide bonds forming an “inhibitory cysteine knot” inhibit

KV4 currents, including Heteropoda venatoria toxins (Sanguinetti et al., 1997), HpTX2 (Zarayskiy et

32 al., 2005), HpTX3 (Brahmajothi et al., 1999), the Phrixotrichus auratus toxins PaTx1 and PaTx2 (Diochot et al., 1999) and the Theraphosa leblondi toxins TLx1-3 (Ebbinghaus et al., 2004). These toxins bind to the extracellular linker region between transmembrane segments S3B and S4 (DeSimone et al., 2009; Zarayskiy et al., 2005) and interfere with the channels gating mechanism resulting in a stabilization of the closed state configuration (DeSimone et al., 2009, 2011). Recently, it has been shown that KV4 channels can be blocked by the AmmTX3 of the α-KTX15 family of scorpion toxins, principally in the presence of the auxiliary subunits DPP6 and DPP10 (Maffie et al.,

2013). Besides spider toxins, KV4 currents and Ito,f can be blocked by 4-aminopyridine (4-AP) in the millimolar range (Wang et al., 1995). However, 4-AP also blocks KV1 channels and Ito,s in the micromolar range. Interestingly, the underlying mechanisms are different; 4-AP binds to Ito,f channels in the closed state and to Ito,s channels in the open-state, resulting in different use- dependence effects (Patel and Campbell, 2005). Ito,f is also blocked by several sodium channel blockers, including flecainide (Wang et al., 1995) and quinidine (Imaizumi and Giles, 1987) as well as several calcium channel blockers, including nifedipine (Bett et al., 2006).

We have recently added to the list of Ito modulating drugs by identifying two bis-phenyl urea compounds, NS5806 (Calloe et al., 2008) and NS3623 (Calloe et al., 2016). Both compounds increase

Ito,f. NS5806 has been suggested to modulate the interaction between the hydrophobic groove on 2+ KChIP2 and KV4 as well as to affect Ca sensitivity of KChIP2 (Gonzalez et al., 2014); however, in the absence of KChIP2, NS5806 still modulates KV4.3 currents (Lundby et al., 2010) suggesting additional binding sites for NS5806 on KV4 α subunits. NS3623 was initially described as a chloride channel blocker (Bennekou et al., 2001) and was later found to activate IKr by slowing the onset of inactivation (Hansen et al., 2006, 2007, 2008). However, NS3623 is also a potent activator of Ito,f in the same micromolar ranges as required for IKr activation (Calloe et al., 2016).

7.3.3 The effect of Ito activators NS5806 and NS3623 on isolated canine ventricular cells The magnitude of the early repolarization is the result of a balance between depolarizing and repolarizing currents. NS5806 increases the early repolarization in canine epicardial cells (Figure 15) (Calloe et al., 2008). The increase in early repolarization affects APD in canine hearts resulting in two possible outcomes: i) A deep "notch" and a delay of the action potential dome. This is associates with a marked prolongation of the APD. ii) Alternatively, the early repolarizing overpowers ICaL resulting in "loss-of-the-dome" and dramatically abbreviated action potentials, reminiscent of those found in small rodents (Figure 15B) (Calloe et al., 2008; Giudicessi et al., 2011; Grubb et al., 2014;

Sah et al., 2003). In rodents and lagomorphs where Ito,f contributes to late repolarization, increased

Ito,f results in a shortening of the action potential (Cheng et al., 2017). Thus, the effect of increasing

Ito,f on action potential duration is species dependent.

In isolated canine midmyocardial cells NS5806 increases Ito,f peak currents and slows current decay

(Figure 15C-E) resulting in a large increase in total charge carried by Ito (Figure 15F).

Figure 15: The Ito,f activator NS5806. (a) Chemical structure of the diphenylurea 1-(3,5-Bis-trifluoromethyl-phenyl)-3- [2,4-dibromo-6-(1H-tetrazol-5-yl)-phenyl]-urea compound NS5806. (b) Action potentials recorded from an isolated canine LV Epi myocyte before (black) and at 6, 10 and 14 s after application of 10 μM NS5806 (grey) at a BCL of 2000 ms. (c) Representative Ito recordings from isolated LV Mid cells in the absence and presence of 10 μM NS5806. (d) Current-voltage (I-V) relation of peak Ito, (e) time constant of decay of Ito and (f) area under the curve, reflecting the total charge carried by Ito in absence and presence of 10 μM NS5806, n=7. Reprinted from (Calloe et al., 2008) with permission from Oxford University Press.

The increase in Ito is only found in mid- and epicardial cells, where NS5806 increases Ito density by approximately 80 % at +40 mV. In endocardial cells where Ito density is low, the effect of NS5806 is not significant (Figure 16). Application of NS5806 results in a significant left shift of the voltage- dependence of steady-state inactivation in all three cell layers (Figure 17). This shift in steady-state inactivation would tend to decrease currents, highlighting the complex and multiple effects of

NS5806 on Ito gating.

Figure 16: NS5806 increases Ito in Mid- and Epi but not in Endo cells. (a) Representative Ito recordings from isolated canine LV Epi and (b) Endo cells under control conditions and in the presence of NS5806 (10 μM). Cd2+ was present to block ICa. (C) The current-voltage relation for peak Ito from Epi (d) Mid and (e) Endo cells in the absence and presence of NS5806 (10 μM). Reprinted from (Calloe et al., 2010) with permission from Elsevier.

Figure 17: NS5806 induce a hyperpolarizing shift in Ito steady-state inactivation. Ito was recorded from isolated canine 2+ LV cells in absence or presence of NS5806 (10 μM). Cd was present to block ICa. (a) Representative traces recorded from a Mid cell under control conditions and (b) after application of NS5806. Voltage-dependence of inactivation was determined by fitting Boltzmann curves to the normalized currents at the +20 mV step for (c) Epi, (d) Mid and (e) Endo cells. In control the mid-inactivation voltages (V1/2) were -46.0 ± 1.0 mV for Epi, -43.7 ± 0.8 mV for Mid, and -52.9 ± 1.1 mV for Endo cells. In presence of NS5806 (10 µM) V1/2 was -52.0 ± 0.9 mV for Epi, -48.7 ± 0.7 mV for Mid and -56.3 ± 0.8 mV for Endo cells. Reprinted from (Calloe et al., 2010) with permission from Elsevier.

35 In isolated canine mid- and epicardial myocytes, the time-dependent recovery from inactivation of

Ito follows a bi-exponential course. NS5806 accelerates recovery from inactivation and in the presence of NS5806 the recovered current follows a mono-exponential time course (Figure 18).

There is no significant effects of NS5806 on recovery of Endo Ito (Calloe et al., 2010).

Figure 18: NS5806 accelerates time-dependent recovery from inactivation. (a) Representative Ito traces recorded from an isolated canine LV midmyocardial cell under control conditions and (b) after application of NS5806 (10 μM). The normalized recovery time course as a function of time in the absence and presence of 10 μM NS5806 for (c) Epi, (d) Mid 2+ and (e) Endo. Cd was present to block ICa. There was a fast and a slow phase of recovery as follows: 1 = 43.8 ± 6.7 ms and 2 = 256.6 ± 18.9 ms for Epi cells, 1 = 39.7 ± 8.3 ms and 2 = 278.4 ± 19.2 ms for Mid, and 1 = 75.2 ± 8.6 ms and 2 = 803.9 ± 79.8 ms for Endo. In the presence of NS5806, the reactivation of Ito in Epi and Mid cells could be fit with a single exponential with  = 55.6 ± 1.7 ms for Epi and  = 71.3 ± 2.8 ms for Mid. The reactivation time course of Ito in Endo cells was unchanged with 1 = 87.2 ± 8.2 ms and 2 = 705.6 ± 81.8 ms. Reprinted from (Calloe et al., 2010) with permission from Elsevier.

Surprisingly, NS5806 slows recovery from inactivation of KV4.3 channels expressed in heterologous systems and co-expression of KV4.3 with different regulatory β subunits does not recapitulate native

Ito with regard to the effects of NS5806 (Lundby et al., 2010). This prompted us to investigate the effect on other ion channels (Calloe et al., 2008, 2011a; Lundby et al., 2010). Interestingly, KV1.4 channels are inhibited by NS5806. This suggest that the acceleration of recovery from inactivation observed in canine mid- and epicardial myocytes is because of concomitant enhancement of the fast recovering Ito,f and block of the slowly recovering Ito,s, in agreement with a shift from a bi- exponential to a mono-exponential time course of recovery. This is further supported by experiments where KV4.3 + KChIP2 + DPP6 are co-expressed with KV1.4 channels in Xenopus laevis oocytes (Lundby et al., 2010).

36 7.4 Pathophysiological implications of Ito,f

7.4.1 Ito,f and cardiac arrhythmias

Ito,f has been implicated in several pathophysiological conditions and inherited syndromes, including the Brugada syndrome (BrS). The BrS is a condition associated with an increased risk of ventricular fibrillation and sudden cardiac death, often triggered by fever or increased vagal tone. The prevalence of the BrS is estimated at 1–5 per 10,000 inhabitants worldwide. Males are more commonly affected than females. The BrS is characterized by elevated J waves or ST segments in the right precordial leads in the absence of structural heart disease (Brugada and Brugada, 1992). However, the underlying electrophysiological mechanism(s) of this defining feature is still being debated. Two hypotheses have been proposed (Figure 19): The depolarization hypothesis relies on right ventricular conduction slowing and involvement of (mild) structural abnormalities. The conduction slowing results in a delayed activation of the right ventricular outflow tract (RVOT) compared to other regions of the right ventricle (RV) and the resulting electrical gradient is reflected on V2 leads as an ST segment elevation (Figure 19, Left Panel). The repolarization hypothesis relies on an imbalance between depolarizing and repolarizing currents in the early part of the action potential. If INa is decreased or Ito increased, the early repolarization may overpower ICaL which may result in loss-of-the-dome of action potentials in mid- and epicardial cells. Because Ito,f is not expressed in endocardial cells, the action potential plateau is preserved in those cells. The resulting disparity in early repolarization sets the stage for phase 2 re-entry arrhythmias and is suggested to underlie the J wave elevation associated with the BrS. The repolarization hypothesis is supported by experimental data from canine ventricular wedge preparations were Ito was increased by NS5806 (Figure 19, Right panel). However, both hypotheses are strongly supported by experimental and clinical evidence (for more details, see Point/Counterpoint Discussion (Wilde et al., 2010)) and the two hypotheses are not mutually exclusive. It is thus possible that both mechanisms contribute to the pathology (Lambiase et al., 2009).

30 % of the BrS cases have been linked to loss-of-function mutations in cardiac INa but in the majority of patients the underlying mechanism remains unresolved (Nielsen et al., 2013). Recently, gain-of- function mutations in KV4.3 have been linked to BrS (Leu450Phe and Gly600Arg (Giudicessi et al., 2011)) and to sudden unexpected death (SUD, Val392Ile and Gly600Arg (Giudicessi et al., 2012)), supporting the repolarization hypothesis (Figure 14 and 19). These mutations enhance KV4.3 currents and computer modelling suggests two possible scenarios: i) An increased magnitude of the early repolarization resulting in elevated J waves and prolonged APD. ii) The early repolarization overpowers ICaL resulting in loss-of-the-dome and abbreviated ventricular action potentials

(Giudicessi et al., 2011). This is in agreement with our experimental data using the Ito,f activator NS5806 in the canine wedge model (Figure 19).

Figure 19: The depolarization versus the repolarization hypothesis for Brugada syndrome. Left: The depolarization hypothesis states that conduction slowing in the right ventricle (RV) results in a delayed activation of the right ventricular outflow tract (RVOT) compared to the RV (A and B). D) During the hatched phase, RV cells are depolarized compared to the RVOT cells, thus RV cells are acting as a source for current flow toward RVOT cells that act as a sink (C,a). The current passes back to the RV in the extracellular space (C,c). This results in a positive signal on ECG leads over the RVOT (e.g. V2), explaining the ST elevation (D, bold line). F) In the next phase (hatched) the potential gradients between the RV and the RVOT are reversed and the membrane potentials are now more positive in the RVOT compared to the RV and currents are now passing away from V2 resulting in a negative T-wave (F, bold line). Reprinted from (Wilde et al., 2010) with permission from Elsevier. Right: The repolarization hypothesis holds that an imbalance between depolarizing and repolarizing currents in the early parts of the action potential results in a transmural dispersion of the early repolarization. A) Different concentrations of the Ito,f activator NS5806 were used to mimic BrS in a canine LV wedge. Epicardial action potentials show an augmented early repolarization and prolonged APD whereas others exhibit loss-of-the-dome morphology and there is little effect on endocardial action potentials, resulting in prominent J waves on the transmural electrocardiogram (ECG). B) Multiple arrhythmic runs in the presence of 15 µM NS5806. The arrows indicate pacing stimuli. Co-existence of short and long APs sets the stage for phase 2 re-entry circuits, where a "dome" excites neighbouring areas where the dome is lost. As the early repolarization is more prominent in RV, the resulting J- wave is most prominent in ECG leads facing the RV (data not shown). The figure is based on data presented in (Calloe et al., 2008).

We have identified a KV4.3 gain-of-function (Ala545Pro) in early onset atrial fibrillation (AF) (Olesen et al., 2013). Atrial action potentials do not exhibit the classical spike-and-dome configuration and modelling studies suggests that in the atria increased Ito,f lowers the voltage of the plateau phase and abbreviates the atrial action potential (Courtemanche et al., 1998; Olesen et al., 2013). The AF

38 patient did not exhibit a BrS ECG phenotype under a flecainide challenge (Olesen et al., 2013) and not all BrS patients with KV4.3 gain-of-function mutations have documented AF (Giudicessi et al., 2011, 2012), suggesting that the expression and/or regulation of other cardiac currents may influence the phenotype.

Since increased early repolarization may play a central role in the BrS, Ito blockers could potentially be of therapeutic value. In support of this, quinidine has been shown to reduce the occurrence of arrhythmias in BrS patients (Márquez et al., 2012). However, quinidine is associated with frequent gastrointestinal side-effects (Nielsen et al., 2013) and development of better Ito antagonists could potentially be beneficial for treating BrS patients.

Besides being expressed in the heart, KV4.3 has important roles in neuronal tissue. Several amino acid substitutions in KV4.3 have been associated with inherited spinocerebellar ataxia (SCA), a progressive disease associated with atrophy of the cerebellum causing ataxia of gait, limbs and eye movement as well as speech impediments (Duarri et al., 2012; Lee et al., 2012; Smets et al., 2015).

The reported SCA KV4.3 mutations exhibit impaired folding and exert a dominant negative effect on trafficking and surface expression of wild-type KV4.3 resulting in neuro-degeneration (Duarri et al., 2012, 2015). Notably, Leu450Phe has been associated with both BrS and SCA (Duarri et al., 2013). This puzzling observation has not been resolved, but electrophysiological studies revealed

Leu450Phe results in a gain-of-function of KV4.3. For the loss-of-function mutations associated with SCA, no cardiac phenotype has been reported to date (Duarri et al., 2012, 2015) and, similarly, no neuronal phenotype have been reported for the BrS or AF associated gain-of function mutations.

7.4.2 Ito,f in heart failure Heart failure (HF) is defined as “a complex clinical syndrome that can result from structural or functional cardiac disorders that impairs the ability of the ventricles to fill or eject blood” (Jessup et al., 2009). Typically HF represents the final stage of various cardiac diseases including ischemia, valvular diseases or hypertension. Diagnosis of HF is based on the presence of symptoms such as dyspnoea, fatigue, limited exercise tolerance and oedema (Roger, 2013). Initially increased sympathetic stimulation compensates for the reduced cardiac output by maintaining blood pressure and cardiac output. However, this increases the metabolic demands of the heart and decreases the cardiac efficacy. During the progression of HF a cascade of events occurs including fibrosis, ultrastructural changes, cellular hypertrophy, loss of T-tubules (Aistrup et al., 2013; Shah et al., 2014) and alterations in Ca2+ cycling proteins (Bers, 2006; Wasserstrom et al., 2009). Normally CaV1.2 channels are predominantly found in the T-tubules positioned opposite to clusters of RyRs in the SR. Disruption of the T-tubular system alters this spatial organization and contributes to a less synchronized CICR (Aistrup et al., 2013; Shah et al., 2014). Decreased expression of SERCA and up-regulation of NCX result in a larger fraction of Ca2+ being pumped out of the cell and a defective sequestration of Ca2+ into the SR (O’Rourke et al., 1999; Pogwizd et al., 1999). The reduced SR Ca2+ loading results in 2+ 2+ smaller and unsynchronized Ca transients, resulting in decreased systolic [Ca ]i. In contrast, 2+ diastolic [Ca ]i is thought to be elevated due to deficient removal (Piacentino et al., 2003). When 2+ the transient amplitude is decreased, increased Ca entry through CaV1.2 during the plateau phase

39 is predicted due to less Ca2+-dependent inactivation further contributing to the increased diastolic 2+ 2+ [Ca ]i (Cooper et al., 2010). Hyper-phosphorylation of RyRs results in Ca leaks. The mechanism(s) behind the hyper-phosphorylation is being debated but increased PKA (Marx et al., 2000) or CaMKII activity (Bers, 2006) have been suggested to play a role. These alterations in Ca2+ handling are thought to play an essential part in the progressive deterioration of cardiac function in heart failure.

Beside changes in ultrastructure and Ca2+ homeostasis, the cellular electrophysiology is also affected by hypertrophy and heart failure. Several of the repolarizing potassium currents are down-regulated including Ito,f and IKr (Beuckelmann et al., 1993; Cordeiro et al., 2012; Janse, 2004; Näbauer and Kääb,

1998; Shah et al., 2014; Zicha et al., 2004). We found a progressive reduction in Ito in isolated canine LV cardiomyocytes in a canine model where hypertrophy was induced by ventricular tachypacing for either two or five weeks (Figure 20). This reduction in Ito was reversed by application of NS5806

(10 µM). In cardiomyoctes from hearts tachypaced for two weeks, Ito was completely restored to normal and in cardiomyocytes from hearts tachypaced for five weeks there was a significant increase in Ito after application of NS5806 (Cordeiro et al., 2012).

The mechanism(s) underlying the down-regulation of Ito,f are not fully understood. Activated calcineurin may act as a physiological regulator of both the cardiac hypertrophic response

(Molkentin, 2000) as well as Ito down-regulation (Perrier et al., 2004). Similarly, CaMKII activity and expression are increased in various animal HF models as well as in human HF (Luo and Anderson,

2013) and CaMKII phosphorylation results in a decreased expression of KV4.3 in canine cardiomyocytes (Xiao et al., 2008). The constitutive sympathetic activation could also play a direct or indirect role in the progression of HF and in the down-regulation of Ito,f (Panama et al., 2011).

Ventricular expression of KChIP2 is decreased in patients with HF (Radicke et al., 2006; Soltysinska et al., 2009) as well as in experimental models of hypertrophy (Jin et al., 2010; Speerschneider et al., 2013). This loss of KChIP2 has been proposed to underlie the decreased Ito and gene transfer of KChIP2 attenuates the development of ventricular hypertrophy in aortic banded rats supporting a regulatory role of KChIP2 in hypertrophic remodelling in rats (Jin et al., 2010). However, down- regulation of KChIP2 is not a consistent finding in HF (Akar, 2005; Goltz et al., 2007; Zicha et al., 2004) and we found that the time course of the progression of hypertrophy is similar in WT and KChIP2 deficient mice (Speerschneider et al., 2013).

Figure 20: Activation of Ito in epicardial cells from failing hearts. Dogs were tachypaced for 2 or 5 weeks to induce HF. (a) Representative Ito from a LV Epi cell from a 5-week tachypaced canine heart under control conditions and (b) after application of 10 μM NS5806. (c) Mean current-voltage (I-V) relation for peak Ito from 2-week and (d) 5-week tachypaced Epi cells in absence and presence of 10 μM NS5806. The time constants of decay (Tau) were obtained by fitting single exponential equations to the decaying phase of the currents and plotted as a function of voltage for (e) 2-week and (f) 5-week tachypaced Epi cells. Ito from normal Epi cells is included in the graphs for comparison. Reprinted from (Cordeiro et al., 2012) with permission from Elsevier.

The reduction in Ito results in a reduced early repolarization of mid- and epicardial action potentials.

The reduced IKr results increased APD in all layers of the ventricular wall in failing hearts (Figure 21). Application of NS5806 resulted in at least a partial restoration of the early repolarization in the mid- and epicardium, which is reflected in the augmented J wave on the transmural ECG. The action potential prolongation associated with HF was unaffected by NS5806.

41 Figure 21: NS5806 normalizes early repolarization of action potentials in failing hearts. Action potentials and transmural ECGs were recorded from canine left ventricular wedges from healthy controls and from HF hearts paced at 2000 ms BCL. HF was induced in canine hearts by 2-weeks of ventricular tachypacing at 220 bpm, resulting in decreased early repolarization in Epi and increased APD in all layers. Recordings from the same HF wedge in absence or presence of 10 µM NS5806 are shown. Representative of n = 3. Reprinted from (Cordeiro et al., 2012) with permission from Elsevier.

In Category A hearts (e.g. human and canine hearts), the loss of Ito,f and early repolarization in HF could impair the contractile function by two different mechanisms: i) Reduced Ito,f and early 2+ repolarization result in decreased ICaL, smaller and dys-synchronized Ca transients at the single cardiomyocyte level. This has elegantly been demonstrated in healthy isolated rat, rabbit and feline ventricular cardiomyocytes where AP clamp using failing human action potentials resulted in depressed transients compared to AP clamp using healthy human action potentials as command

(Cooper et al., 2010; Harris et al., 2005). ii) In Category A hearts, loss of Ito,f results in a reduced transmural gradient of early repolarization as shown for canine hearts in Figure 20 (Cordeiro et al., 2012). As the transmural gradient in early repolarization may be important to synchronize Ca2+ transients and cell shortening across the ventricular wall, we hypothesise that loss of Ito,f affects the synchronization of transmural contraction and worsen energy expenditure in the failing heart.

Yet, the effects of reduced Ito,f on transmural coordination of contraction in failing hearts have not been thoroughly investigated. A study on human wedge preparations from normal and failing explanted hearts using voltage sensitive dyes demonstrated a tendency towards an increased delay from action potential upstroke to Ca2+ transients, however, the effect was most pronounced in the endocardium (Lou et al., 2011). Thus, these experiments do not fully support this hypothesis.

7.5 Is pharmaceutically increased Ito,f potentially beneficial in failing hearts?

Augmentation of Ito,f in heart failure may have beneficial effects on cardiac output as restoration of the spike-and-dome morphology will result in an immediate increase in Ca2+ influx through L-type Ca2+ channels. This is thought to improve calcium transients and contraction at the cellular level as elegantly demonstrated by AP clamp experiments by Copper et al., and Harris et al., (Cooper et al., 2010; Harris et al., 2005). Improved Ca2+ influx will also help to maintain SR Ca2+ load since a greater amount of Ca2+ will enter the cell during each action potential. We have shown that pharmacological

42 enhancement of Ito,f re-establishes the transmural gradient of early repolarization (Cordeiro et al., 2012). The restoration of the transmural gradient of early repolarization may improve the synchronization of contraction across the ventricular wall and thereby reduce the stress of the failing heart. Based on the observations hypothesis 2 is proposed:

Restoration of Ito,f in the setting of heart failure has several beneficial effects: i) Restoration of Ito,f increases Ca2+ influx and Ca2+ transients and thereby improve contractility at the cellular level. ii) By restoring Ito,f, the transmural gradient in early repolarization is restored resulting in improve transmural coordination of Ca2+ transients and contraction. This decreases the energy expenditure of the heart and improves cardiac efficacy.

While it is evident that NS5806 restores the spike-and-dome morphology in action potentials from canine wedge preparations from failing hearts, the R wave remains wide, reflecting hypertrophy and/or decreased conduction velocity (Figure 21) (Cordeiro et al., 2012). Furthermore, the AP prolongation is unaffected or even aggravated by NS5806 (Figure 19). Prolonged APD is associated with a high risk of early after-depolarization (EAD) that may trigger arrhythmias (Bers, 2006). The 2+ EADs are thought to be the result of reactivation of ICaL which has recovered from Ca induced inactivation. Approximately 50 % of HF patients die from cardiac arrhythmias and indeed, these arrhythmias are typically triggered by EADs or delayed after-depolarizations (DADs). The DADs are initiated by spontaneous SR releases of Ca2+ activating the electrogenic NCX (Aistrup et al., 2011).

This led us to investigate the effects of NS3623 that enhances both Ito and IKr on cultured canine cardiomyocytes. Previous studies have shown that ventricular cells in culture undergo electrical remodelling resulting in a reduced repolarization reserve (Roden, 1998), including a reduction of Ito, IKr, and ICa (Louch et al., 2004). In agreement with these results, we found that Ito and IKr in LV canine midmyocardial cells are reduced after 24 hours in culture and action potentials exhibit decreased early repolarization and prolonged duration associated with an increased tendency for EADs (Figure 22) (Calloe et al.,

2016). Similarly to NS5806, NS3623 slows canine midmyocardial Ito decay, shift the steady-state inactivation towards more negative potentials and accelerates recovery from inactivation (Calloe et al., 2016). The effects on action potential morphology in canine wedges are also similar to those of

NS5806, except the concomitant enhancement of IKr prevents the prolongation of the APD that is associated with activation of Ito (Calloe et al., 2016). Application of NS3623 to cultured cardiomyocytes results in a partly restoration the early depolarization, a significant shortening of the APD and suppression of EADs (Figure 22) (Calloe et al., 2016).

These experiments suggest that there is potential benefit of modulation of Ito,f as well as IKr under conditions were the repolarization reserve is reduced, such as hypertrophy and heart failure.

43 Figure 22: The effect of NS3623 on cultured canine midmyocardial cells. (a) Representative AP recordings from a freshly isolated LV Mid cell (b) AP recording from a LV Mid cell kept in culture for 1 day or (c) for 2 days. Cells were paced at BCL = 1 s (a, c and e) or at BCL = 2 s (b and d). Recordings from cultured cells were in absence and presence of 5 µM NS3623. Representative of ≥n = 4 cells from N = 3. Reprinted from (Calloe et al., 2016) with permission from Elsevier.

44 8 CONCLUDING REMARKS AND FUTURE PERSPECTIVES

Despite substantial differences in cardiac electrophysiology animal hearts are extensively used as models for human hearts when investigating mechanisms underlying cardiac diseases as well as in safety pharmacology (Hamlin, 2005). These differences may have serious implications if research results are extrapolated from animals to humans. For instance, rodents and lagomorphs have fast heart rates and concomitant short APDs. In these species Ito contributes to the late repolarization phase, thus, the effects of manipulating Ito are not comparable to those in human hearts (Sah et al., 2003), and precaution is warranted for many other currents. Canine and porcine hearts have been proposed to serve as good models for human hearts; the hearts are equal size, they weigh the same, in the porcine heart the coronary circulation and lack of collaterals are similar to in human hearts, whereas canine hearts have extended collateral perfusion. However, unlike human hearts, porcine hearts have Purkinje fibres that penetrate the ventricular wall and Ito,f is not present (Schultz et al., 2007). Canine hearts may be a closer match to human hearts with regard to cardiac electrophysiology; the Purkinje fibres are predominantly found in the endocardial layers and there is a large Ito,f in mid- and epicardial layers (Hamlin, 2007, 2010; Jost et al., 2013).

8.1 The physiological significance of the Purkinje fibre distribution Based on our experimental work as well as the work of other groups Hypothesis 1 is proposed:

A coordinated contraction of the endo-, mid-, and epicardial layers of the ventricular walls can be the result of anatomical adaptations, such as deep penetrating Purkinje fibres (e.g. equine and porcine hearts) or regional differences in expression of Ito,f resulting in a transmural gradient in the early repolarization of the ventricular action potentials (human and canine hearts).

The hypothesis is supported by the fact that many Category A hearts exhibits transmural gradient in early repolarization whereas Category B hearts have more homogenous action potential waveforms across the ventricular wall (Table 1). To gain further support for the hypothesis, it would be interesting to investigate the electrophysiology in large carnivore hearts where the delay in epicardial activation in the absence of deep penetrating Purkinje fibres could be substantial. Based on ECG data it appears that even large carnivore hearts, such as brown bear hearts, display similar QRS vectors as other Category A hearts (Gandolf et al., 2010). This suggests that similar to canine and human hearts, the depolarization wave propagates from the endocardium to the epicardium.

It would be interesting to investigate action potential morphology and whether a gradient in Ito and early repolarization across the ventricular wall exist. Would a gradient in early repolarization be sufficient to compensate for the conduction delay from the endo- to the epicardium in very large hearts or are other mechanisms at play?

45 The distribution of the Purkinje network has other interesting implications. It is estimated that in ischemic hearts 60 % of arrhythmias are initiated in the Purkinje fibres (Hirose et al., 2008; Lopera et al., 2004). The relative long APD makes the Purkinje fibres susceptible to EADs and the connections between Purkinje and ventricular cells have a relative low safety margin for retrograde conduction. Differential conduction delays or anterograde block at one junction could provide the substrate for a classical re-entry circuit (Valderrábano et al., 2001). This could explain why the pig develop arrhythmias, including ventricular arrhythmias with little provocation compared to canine hearts (Hamlin, 2007, 2010). This also has implications for using animal hearts as models for human hearts. Arrhythmias may originate from different cardiac regions in Category B hearts compared to Category A hearts and the presence of deep Purkinje fibres may also affect the conduction velocity of re-entry circuits (Allison et al., 2007; Newton et al., 2004).

The different distribution of the Purkinje network as well as the differences in action potential configurations including the lack of Ito,f demonstrate that Category B hearts (e.g. goats, pigs and horses) do not recapitulate human electrophysiology. For instance, many cardiomyopathies, including hypertrophy and heart failure are associated with a reduced Ito in humans. This reduction 2+ in Ito will impact both cellular Ca handling as well as transmural coordination of contraction (Cooper et al., 2010; Cordeiro et al., 2012). Thus, the Category B hearts may be poor models for the human heart in regard to mechanism(s) underlying the progression of hypertrophy and heart failure.

8.2 Is pharmaceutically increased Ito,f potentially beneficial in heart failure? The lifetime risk of developing HF is a staggering 20 % (Lloyd-Jones et al., 2002). The 5 year mortality is 50 %, approximately half dies from arrhythmias, the other half from pump failure (Roger, 2013). 2+ To provide short-term inotropic support for the failing heart, agents that increase [Ca ]i such as adrenergic agonists, type 3 phosphodiesterase inhibitors and blockers of the Na+/K+-pump are used (Braunwald, 2013). These agents can be life-saving in acute HF, but the oxygen consumption is increased resulting in a decreased efficacy of the heart. In contrast, in chronic HF the aim is to reduce the work load of the heart, either by reduction of neurohumoral stimulation by administration of β- blockers or by reducing the afterload by administration of angiotensin converting enzyme inhibitors, angiotensin II inhibitors or diuretics (Braunwald, 2013).

It has convincingly been demonstrated that the presence of an early repolarization results in larger L-type Ca2+ currents, larger and more synchronized transients in isolated failing cardiomyocytes

(Cooper et al., 2010; Harris et al., 2005) and the notion that Ito plays an important role in transmural coordination of contraction (Cordeiro et al., 2004) led to hypothesis 2:

46 transmural coordination of Ca2+ transients and contraction. This decreases the energy expenditure of the heart and improves cardiac efficacy.

We have demonstrated that application of the Ito,f activator NS5806 results in restoration of the spike-and-dome morphology of action potentials from canine failing hearts (Cordeiro et al., 2012). To determine if normalizing action potential shape is beneficial in hypertrophy or heart failure, the AP-clamp technique can be used to provide proof-of-concept by comparing the effects of healthy and failing APs as command potential on Ca2+ transients and contractility in isolated cardiomyocytes from failing hearts. The potential beneficial effect of restoring the transmural gradient of early repolarization on synchronization of contraction across the ventricular wall in failing Category A hearts also needs experimental support. Currently the experimental work is hampered by the lack of specific Ito,f activators and hopefully novel Ito,f activators or inhibitors as well as the greater technological potential for viral gene transfer will greatly enhance the prospects of exploring the effects of Ito,f activators in the future.

Finally, it is important to note that hypertrophy and heart failure are complex syndromes and often represent the final stage of other cardiac diseases, with hypertensive and ischemic heart diseases being the most common (Aistrup et al., 2011). During the progression of heart failure many other alterations take place, including changes in Ca2+ handling, ultra structural changes and fibrosis.

Restoration of Ito may not improve these structural changes and obviously, it is important to treat the comorbidities. However, augmentation of Ito,f may improve the contractile function of the heart both at the cellular level by restoring Ca2+ transients and by synchronizing contraction across the ventricular wall. This could potentially minimize the stress of the failing heart and increase cardiac output without increasing energy expenditure.

8.3 Methological considerations The level of observed heterogeneity across the ventricular wall is influenced by recording method. In intact tissue, the intrinsic APD heterogeneity can be limited by electrotonic interactions between cells; cells that depolarize later will generate repolarizing electrotonic currents to neighbouring depolarized cells (Boukens et al., 2017; Laurita et al., 1997), thus coupling tends to attenuate the electrical dispersion in the heart and the differences in action potentials will be augmented in isolated cells. In vivo, cardiomyocytes interact with neighbouring cells, and are under normal circumstances never isolated, which merit the use of the wedge preparations. On other hand, the wedge preparation is characterized by extensive cut surfaces and perfusion may not be evenly distributed (Wilson et al., 2009). Recordings using floating sharp electrodes give unstable impalements and cannot reliably measure membrane potential, however, it gives an accurately measure of the ADP. On the other hand, the use of voltage sensitive dyes can also affect the electrophysiological properties, often blebbistatin is used to immobilize the tissue may affect

47 cellular electrophysiology as well as Ca2+ transients (Brack et al., 2013) and the time resolution is poor.

8.4 Conclusion Human hearts are examples of Category A hearts that are characterized by a depolarization that spreads through the endocardium from the apex to the free walls, and then from the endocardium to the epicardium. In healthy hearts Ito,f plays an important in synchronizing transmural contraction, however, many cardiac pathologies including hypertrophy and heart failure are associated with a loss of Ito,f and the transmural gradient in early repolarization. Pharmacological restoration of Ito,f in the setting of heart failure may improve Ca2+ transients and contraction at the cellular level as well as across the ventricular wall and thereby decrease the energy expenditure of the heart and improve cardiac efficacy. As increased Ito,f is associated with a risk of phase 2 re-entry arrhythmias as associated with the Brugada syndrome, it important to determine a “Goldilock zone” where the beneficial effects are present, yet, without hampering cardiac safety.

9 ACKNOWLEDGEMENTS

The presented work has been supported by the Arvid Nilssons Fond, the Carlsberg Foundation, Brødrene Hartmanns Fond, the Danish Cardiovascular Research Academy (DaCRA), Kong Christian den Tiendes Fond, Novo Nordisk Foundation, Eva og Henry Frænkels Mindefond, Fonden af 17.12.1981, Margrethe Møller Fonden, Etly og Jørgen Stjerngrens fond, Christian og Ottilia Brorsons Rejselegat for yngre videnskabsmænd og kvinder, Carl og Ellen Hertzs Legat til Dansk Læge- og Naturvidenskab.

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