Doctoral Dissertation

Abstract

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. 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-H75. 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. *Contributed equally. J Mol Cell Cardiol. 2012 Jun;52(6):1291-1298. 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):1057-1066. 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):2028-2044. 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):686-694. Editorial comment: in Cardiovasc. Res. 2009, 81:635- 636. The present thesis is based on studies performed at the Department of Veterinary and Animal Science (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. None. 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. 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). Hypotese 2: Farmakologisk forøgelse af Ito,f under hjertesvigt har flere gavnlige effekter: (a) I den enkelte hjertecelle vil forøget Ito,f øge ICaL hvorved Ca2+-transienterne normaliseres og kontraktiliteten forbedres. (b) 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. 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 (eg, 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 (eg, human and canine hearts). Hypothesis 2: Restoration of Ito,f in the setting of heart failure has several beneficial effects: (a) Restoration of Ito,f increases Ca2+ influx and Ca2+ transients at the cellular level and thereby improve contractility. (b) 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. 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; 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. Table 1. KChIP2 DPP6 KCNEx KCNJ3 KCNJ5 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 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 are termed IKv4.3+KChIP2 or KV4.3+ KChIP2 current. For canine wedge recordings7 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. 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 (eg, 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 (eg, human and canine hearts). Hypothesis 2: Restoration of Ito,f in the setting of heart failure has several beneficial effects: (a) Restoration of Ito,f increases Ca2+ influx and Ca2+ transients at the cellular level and thereby improve contractility. (b) 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. 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). In addition, precordial leads (V1-V6) are often added, resulting in the 12 lead ECG. A depolarizing wave moving towards 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 ventricles.†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 . 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. 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.9 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 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: (a) It provides a conduction delay between the atria and the ventricles. This allows the atrial systole to take place before the ventricular systole. (b) The AVN has a relatively long refractory period which protects the ventricles from atrial tachyarrhythmias and finally (c) the AVN can serve as a pacemaker because of the 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.9-11 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. 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.12 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.10 Intramural Purkinje fibres have been demonstrated in ungulates including sheep,13 cow,10 pig,12 horse14 and whale.15 In contrast, no intramural fibres have been found in dog, mouse or human hearts.10, 12, 16 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.12 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).17, 18 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). 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.14, 19 Based on the cytoachitecture of the Purkinje fibres and network, a separate Category C for rodents and lagomorphs has been suggested.15, 20 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,21 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 that of canine hearts of equal size19, 22, 23 as the transmural conduction velocity is faster in porcine hearts compared to canine hearts.24 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.22 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 3.19 Although 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.14 The cardiac action potential is because of the orchestrated activation of different ionic currents. It can be divided into 5 phases: Phase 0, the depolarization because of the activation of a rapid sodium Phase the early repolarization because of the activation of transient outward potassium Phase the resulting from activation of calcium current as well the of a or late sodium Phase the late repolarization because of the activation of rectifying potassium current and rectifying potassium currents and Phase the where rectifying potassium currents and potassium channels the potential to the potential for all are present in all cardiac and there are marked between small animals such as have heart of per the action potentials are and without a defined resulting in an of the early and the late repolarization like dogs and have heart the action potentials are and have Action potential in different regions of the heart expression of ion channels and (Figure Action potentials in the heart are of that form This electrical of the cells the heart work as a and will to differences in electrical potential between The action potentials of cells and are different from in atrial or ventricular cells (Figure The potential is to and exhibits a spontaneous depolarization the pacemaker potential which for the intrinsic pacemaker The pacemaker activity is at because of the activation of the current but of calcium from the SR, the also a is by activated channels that of both and In human pacemaker cells, is the the potential to the for activation of voltage calcium the depolarization is mediated by calcium the action potential has a slow velocity and the amplitude is and type Ca2+ channels are present in the SAN. ICaL is for the of the action potential and is by In contrast, ventricular ICaL is by at negative potentials compared to and this may be in pacemaker cells. calcium channels to the late of the The repolarization is because of the of the calcium channels and activation of the potassium currents and mediated by the and channels The acetylcholine activated inward rectifying current by the of the cells and is important for of cardiac Section The inward rectifying potassium current mediated by channels is from tissue. of the is important that the SAN and AVN are electrically insulated from the atrial myocardium. Differential expression of junction is for this The central are of the large and and that are for efficient in the rather the small are expressed in This results in a relatively weak of the cells. the of the SAN the electrical improves with expression of both and of the depolarization from SAN cells to atrial cells through results in the activation of sodium currents by voltage sodium This results in a rapid of the atrial action potential (Figure but the depolarization Ito,f by and Ito,s by KV1.4 as well as the current mediated by resulting in an early The rapid depolarization also ICaL by channels that the depolarization during the atrial to ventricular cells, the atrial potential is depolarized. Atrial action potentials are often as and in however, atrial action potentials recorded from have a and a comparable to ventricular action The late repolarization is a result of of and a activation of voltage potassium including the rectifying and The inward current to the late of repolarization of the action potential and is important for setting the expression is very in the atria compared to the ventricles resulting in a potential of compared to in the In contrast, is large in atrial cells where the cellular is from ventricular Other including the small potassium current mediated by the potassium channels and as well as different also to the a result of expression of the potassium the action potential shows some in different of the From the atria the depolarization wave reaches the The action potential configuration is of action potentials in the SAN, however, the velocity is faster the potential is a and the of the spontaneous depolarization is compared to The expression of ion channels is similar to the SAN, including expression of ICaL by rather than and of and than the small junction is The expression of and the absence of a result in a slow conduction velocity of 5 through the The conduction delay in the AVN the atrial systole to before the ventricular systole. the node the electrical impulses the bundle branches and the system (Figure The Purkinje fibres are for rapid conduction which is reflected in the expression of both the and and factor to the fast conduction is large currents resulting in a fast velocity of the The potential is very negative the The is followed by a repolarization because of the rapid of and activation of The is compared to ventricular as a result of a ICaL The early repolarization and the amplitude of At fast the early repolarization is as there is for Ito to from resulting in a positive This in results in a amplitude of and a of the action potential At slow Ito is large and is small resulting in action potentials. This Ito a role in of the In Purkinje fibres have compared to ventricular because of expression of and A spontaneous 4 depolarization is found in Purkinje fibres to the bundle however, in free running Purkinje from canine ventricles the potential is to the atria the potential of ventricular cells is (Figure The action potentials have a fast velocity a expression of of and activation of Ito,f by with the KChIP2 the early repolarization resulting in a of action potentials in mid- and epicardial In large Ito to the and late repolarization because of its rapid in such as and with action potentials Ito a major late The is of the activation of the channel The repolarization is a result of of and the activation of and and finally The is by properties of these but the activation is with a rapid This is first the potential to at the of the to the late is rectifying resulting in current during the to the late the potential to the potential for In many the action potential configuration differs in different regions of the ventricles (Figure This will be in in Section In the ventricle the of the Ca2+ channels are located in the facing of in the SR (Figure These may than The influx of Ca2+ through Ca2+ channels during the these and a much Ca2+ from the SR, this process has been the Ca2+ induced Ca2+ In of the in Ca2+ is because of the influx through Ca2+ The of Ca2+ from the SR and Ca2+ the the free Ca2+ from a level of to a level of 1 The of Ca2+ from a single is to be the underlying in cardiac of in a results in a Ca2+ The Ca2+ transient represents the and of many Ca2+ the Ca2+ Ca2+ to C, the for on the the and the cells activation of Ca2+ other of Ca2+ can of Ca2+ current or sodium calcium in are also of SR Ca2+ and cellular however, the of these of the has been For