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 phase because of the activation of a rapid sodium current; Phase 1, the early repolarization phase because of the 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 because of 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 resting heart rates, 70-120 bpm, 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.25, 26 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 because of the activation of the “funny” current (If) but cyclic release of calcium from the SR, the “calcium clock” also plays a role.27 If is carried by hyperpolarization activated cyclic-nucleotide gated (HCN) channels that permits passage of both Na+ and K+.28 In human pacemaker cells, HCN4 is the predominant subtype.29 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 CaV1.230 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.31 The repolarization is because of the inactivation of the calcium channels and activation of the delayed rectifier potassium currents IKr (Rapid) and IKs (Slow),32, 33 mediated by the KV11.134 and KV7.1+ KCNE1 channels respectively.35, 36 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 6.6.37, 38 The inward rectifying potassium current IK1 mediated by Kir2 channels is absent from nodal tissue. Because of 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,10, 11 rather the small conductance Cx45 are expressed in nodal tissue.39 This results in a relatively weak coupling of the nodal cells. Towards the periphery of the SAN the electrical coupling improves with expression of both Cx45 and Cx43.39 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 but the depolarization activates Ito,f carried by KV4.2/3 and Ito,s carried by KV1.4 channels40 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.40 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.38, 41, 42 In contrast, IK,Ach is large in atrial cells where it determines the cellular excitability. IK,Ach is absent from ventricular cells.37, 38 Other currents, including the small conductance potassium current mediated by SK channels,43 KV3 channels, the two-pore-domain potassium leak channels TWIK and TASK44 as well as different pumps also contribute to the membrane potential.30 As a result of differential expression of the repolarizing potassium channels, the action potential shape shows some heterogeneity in different areas of the atria.42, 45 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.42 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.30 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.30 The conduction delay in the AVN permits the atrial systole to occur before the ventricular systole. 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),30 which is reflected in the abundant expression of both the large- and intermediate-conductance gap junctions, Cx40 and Cx43. 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).42, 46 The resting membrane potential is very negative (approximately −90 mV), increasing the available INa. The upstroke is followed by a repolarization because of the rapid inactivation of INa and activation of Ito.47 The plateau phase is less depolarized compared to ventricular cells,47 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).47 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,38 possibly because of lower expression of IKr, IKs and IK1.48 A spontaneous phase 4 depolarization is found in Purkinje fibres proximal to the bundle branches,49 however, in free running Purkinje strands from canine ventricles the resting membrane potential is stable.38 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.40, 42 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.40, 50, 51 In large animals, Ito contributes little to the plateau phase and late repolarization because of its rapid inactivation kinetics. However, in smaller animals, such as mice and rats, with short action potentials Ito constitutes a major late repolarizing current.52 The plateau phase is beause of the activation of the L-type channel CaV1.2. The repolarization is a result of inactivation of CaV1.2 and the activation of IKr and IKs and finally IK1.30 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.38, 41, 44 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 7.5. 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).53 In humans, approximately 30% of the increase in intracellular Ca2+ is because of the 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 10 times; from a diastolic level of 100 nmol/L to a peak systolic level of 1 μmol/L.53 The release of Ca2+ from a single RyR is believed to be the elementary event underlying excitation-contraction (EC) coupling in cardiac muscle.54 Activation of 6-20 RyRs in a cluster results in a Ca2+ spark.55 The Ca2+ transient represents the spatial and temporal summation of many Ca2+ sparks.56, 57 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. 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,58 however, the physiological relevance of these alternative triggers of the CICR has been questioned.53 For re