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Introduction to Cardiac Arrhythmias. Electrophysiology of Heart, Action Potential and Membrane Currents

Norbert Jost, PhD

Department of Pharmacology & Pharmacotherapy, Faculty of Medicine,

University of Szeged, Hungary

Correspondence:

Norbert Jost, PhD

Department of Pharmacology & Pharmacotherapy, Faculty of Medicine,

University of Szeged

Dóm tér 12, P.O. Box 427, H-6701 Szeged, Hungary

Tel.: (36-62) 546885, Fax: (36-62) 545680

E-mail: jost.norbert@gmail.com

@All rights reserved to Dr. Norbert Jost, associate professor Department of Pharmacology & Pharmacotherapy, Faculty of Medicine, University of Szeged, Hungary

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Keywords: cardiac membrane potential, action potential, electrophysiology, transmembrane ion channels, List of abbreviations: AF = atrial fibrillation APD = action potential duration AVN = atrioventricular node cAMP= cyclic adenosine monophosphate EK, ENa = equilibrium potential for K+ or Na+, respectively EM= resting membrane potential HVA and LVA = high and low voltage-activated calcium channels,

respectively ICaL= L-type calcium current IK(ACh) = acetylcholine sensitive potassium current IKATP = ATP sensitive potassium current IK = delayed rectifier potassium current; IKr = rapid component of the delayed rectifier potassium current; IKs = slow component of the delayed rectifier potassium current; IK1= inward rectifier potassium current IKur = ultra-rapid component of the delayed rectifier potassium current; INCX = Na+/Ca2+ exchanger current Ito= transient outward potassium current; INa= fast sodium current INaL= late sodium current KV = voltage gated K+ channels LQT3 = long QT3 syndrome mRNA= messenger RNA NaV = voltage gated Na+ channels Na/K pump= sodium-potassium pump NCX= sodium-calcium exchanger current SAN = sinoatrial node Vm = membrane potential

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Table of contents

Abstract

1. Introduction

2. Cell membrane potentials. Electrical activity in the heart: conduction

system and cardiac action potential

2.1. Equilibrium potentials

2.2. Cardiac conduction system

2.3. The cardiac action potential

3. Transmembrane ion channels in the heart

3.1. Voltage-gated Na+ (NaV) currents

3.2. Voltage-gated Ca2+ (CaV) currents

3.3. Voltage gated K+ (KV) channels

3.3.1. Transient outward KV currents (Ito)

3.3.2. Delayed rectifier KV currents (IK)

3.3.3. Inward rectifier potassium currents (IK1)

3.3.3. ATP sensitive potassium currents (IKATP)

3.3.5. Background potassium channels

3.4. Summary

4. Concluding remarks

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Abstract

Myocytes represent the functional unit of cardiac muscle; nonetheless, the heart behaves more or less like an electrical syncytium, whose global activity depends on low resistance coupling between the myocytes. The term “more or less” is used here intentionally to imply that, while the activity intrinsic to individual myocytes is affected by coupling, its features remain recognizable within the context of the whole heart and are important to determine its function. Electrical changes within the myocytes plays an important role to initiate the cardiac contraction. This chapter addresses (a) the electrical activity of individual myocytes, namely the resting membrane potentials and action potentials; (b) the way action potentials are conducted throughout the heart to initiate coordinated contraction of the entire heart; and (c) the transmembrane ionic currents underlying cardiac action potential. 1. Introduction The heartbeat arises from organized flow of ionic currents through ion-specific channels in the cell membrane, through the myoplasm and gap junctions that connect cells, and through the extracellular space. The action potential formation results from the opening and closing (gating mechanism) of several inward and outward ion channels, which are largely expressed within the sarcolemma of cardiomyocytes. Ion channels possess distinct genetic, molecular, pharmacologic, and gating properties, while exhibit heterogeneous expression levels within different cardiac regions. By gating, ion channels permit ion currents across the sarcolemma, thereby creating the different repetitive phases of the action potential, which will be discussed later in detail (e.g., resting phase depolarization repolarization circles). The importance of ion channels in maintaining normal heart rhythm is reflected by the increased incidence of arrhythmias in inherited diseases that are associated to several mutations in genes encoding cardiac ion channels or pumps/exchangers or their accessory proteins and in acquired diseases that are associated with changes in ion channel expression levels or gating properties (e.g., different forms of electrical, structural or contractile remodelling linked to congestive heart failure, dilated cardiomyopathy, permanent forms of atrial fibrillation, etc.). To understand the functioning

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of the transmembrane ion currents and their contribution to the cardiac action potential, it is important to understand the biophysics of the biological cell membranes, including the ion transports, membrane and Nernst potentials as well. 2. Cell membrane potentials. Electrical activity in the heart: conduction system and cardiac action potential Cardiac cells, similar with the majority of the living cells from the body, have an electrical potential across the cell membrane. This potential can be investigated by inserting a microelectrode into the cell and to determine the electrical potential in millivolts (mV) inside the cell relative to the outside of the cell. By convention, the outside of the cell is considered 0 mV. If measurements are taken with a resting ventricular myocyte, a membrane potential of about –90 mV will be recorded. This resting membrane potential (EM) is determined by the concentrations of positively and negatively charged ions across the cell membrane, the relative permeability of the cell membrane to these ions, and the ionic pumps that transport ions across the cell membrane. 2.1. Equilibrium potentials Of several others ions present inside and outside of cells, the concentrations of Na+, K+, Cl-, and Ca2+ are most important in determining the membrane potential across the cell membrane. Table 1 shows typical concentrations of these ions. Among these ions, K+ is the most important in determining the resting membrane potential. In a cardiac cell, the concentration of K+ is relatively high inside (about 140-150 mM), while it is significantly lower outside (4-4.5 mM) the cell. Table 1. Ion concentrations inside and outside of resting myocytes

ION INSIDE (mM) Outside (mM) Na+ 20 145 K+ 150 4

Ca2+ 0.0001 2.5 Cl- 25 140

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Therefore, a strong chemical gradient (concentration difference) exists for K+ that facilitates the ion diffusion out of the cell. The opposite situation is found for Na+; its chemical gradient favours an inward diffusion. The concentration differences across the cell membrane for these and other ions are determined by the activity of energy-dependent ionic pumps and the presence of impermeable, negatively charged proteins within the cell that affect the passive distribution of cations and anions. These concentrations are approximations and are used to illustrate the concepts of chemical gradients and membrane resting potential. To understand how concentration gradients of ions across a cell membrane affect membrane potential, consider a cell in which K+ is the only ion other than the large negatively charged proteins inside of the cell. In this cell, K+ diffuses down its chemical gradient out of the cell because its concentration is much higher inside than outside the cell. As K+ diffuses out of the cell, it leaves behind negatively charged proteins, thereby creating a separation of charge and a potential difference across the membrane (leaving it negative inside the cell).

The membrane potential that is necessary to oppose the movement of K+ down its concentration gradient is termed the equilibrium potential for K+ (EK), and is expressed by the Nernst potential. The Nernst potential for K+ at 37°C is as follows:

mVKKE

o

iK 96

][][log61

where the potassium concentration inside [K+]i = 150 mM and the potassium concentration outside [K+]o = 4 mM. The –61 is derived from RT/zF, in which R is the universal gas constant, z is the number of ion charges (z=1 for K+; z=2 for divalent ions such as Ca2+), F is Faraday’s constant, and T is absolute temperature (°K). The equilibrium potential is the potential difference across the membrane required to maintain the concentration gradient across the membrane. In other words, the equilibrium potential for K+ represents the electrical potential necessary to keep K+ from diffusing down its chemical gradient and out of the cell. An increase in the outside K+ concentration will reduce the chemical gradient for diffusion out of the cell, i.e. the membrane potential required to maintain electrochemical equilibrium would be less negative according to the Nernst equation. The EM for a ventricular myocyte is about –90 mV, which is very close to the equilibrium potential for K+. Because the equilibrium potential for K+ is –96 mV and the resting membrane

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potential is –90 mV, a net driving force (net electrochemical force) acts on the K+, causing it to diffuse out of the cell. In the case of K+, this net electrochemical driving force is the EM (–90 mV) minus the EK (–96 mV), resulting in +6 mV. Because the resting cell has a finite permeability to K+ and a small net outward driving force is acting on K+, K+ slowly leaks outward from the cell. The sodium ions play a major role in determining the membrane potential. Because the Na concentration is higher outside the cell, this ion would diffuse down its chemical gradient into the cell. To prevent this inward flux of Na, a large positive charge is needed inside the cell (relative to the outside) to balance out the chemical diffusion forces. This potential is called the equilibrium potential for Na+ (ENa) and is calculated using the Nernst equation, as follows:

mVNaNaE

o

iNa 52

][][log61

where the sodium concentration inside [Na+]i =20 mM and the sodium concentration outside [Na+]o =145 mM. The calculated equilibrium potential for sodium indicates that to balance the inward diffusion of Na+ at these intracellular and extracellular concentrations, the cell interior has to be + 52 mV to prevent Na+ from diffusing into the cell. 2.2. The cardiac conduction system The conducting system of the heart consists of group of several cardiac muscle cells and conducting fibres, which are specialized for initiating impulses and conducting them rapidly through the heart (Figure 1). They initiate the normal cardiac cycle and coordinate the contractions of cardiac chambers, first contract both atria, then the ventricles. The conducting system provides the heart its automatic rhythmic beat. For the heart to pump efficiently and the systemic and pulmonary circulations to operate in synchrony, the events in the cardiac cycle must be coordinated. The cardiac impulse originates in the sinoatrial node (SA node), located in the right atrium, which is activated first followed by the left atrium. The general direction of the atrial activation is inferiorly, to the left, and posteriorly. This causes the atria to contract and pump blood from the atria to the ventricles; it is recorded on an EKG as a P wave (Figure 1). The atrial impulse is delayed in the atrioventricular node (AV node) to allow the ventricular chambers to fill, and is then conducted rapidly through the ventricles (the bundle of His, the right and left bundles, and the Purkinje fibres). This causes the ventricles to pump blood out of the

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heart and to the body; it is recorded on an EKG as a QRS complex. Recovery following the cardiac cycle, or repolarization, follows.

Figure 1. Electrical activity in the myocardium. Schematic representation of a human heart with illustration of typical action potential (AP) waveforms recorded in different regions, and their contribution to surface electrocardiogram. This is recorded as a T wave on an EKG. On the microscopic level, the wave of depolarization propagates to adjacent cells via gap junctions located on the intercalated disk. The heart is a functional syncytium (not to be confused with a true "syncytium" in which all the cells are fused together, sharing the same plasma membrane as in skeletal muscle). In a functional syncytium, electrical impulses propagate freely between cells in every direction, so that the myocardium functions as a single contractile unit. This property allows rapid, synchronous depolarization of the myocardium. While advantageous under normal circumstances, this property can be detrimental, as it has potential to allow the propagation of incorrect electrical signals. These gap junctions can close to isolate damaged or dying tissue, as in a myocardial infarction.

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2.3. The cardiac action potential The normal mechanical (pump) function of the mammalian heart depends on proper electrical function [1,2], as reflected in the successive activation of cells in specialized, "pacemaker" regions of the heart and the propagation of activity through the ventricles. Myocardial electrical activity is attributed to the generation of action potentials (AP) in individual cardiac cells, and the normal coordinated electrical functioning of the whole heart is readily detected in surface electrocardiograms (Figure 1). Propagation of the electrical activity and coordination of the electromechanical functioning of the ventricles strongly depend on cellular electrical coupling mediated by gap junctions [3]. The generation of myocardial action potentials reflects the consecutive activation and inactivation of ion channels that conduct depolarizing, inward (Na+ and Ca2+), and repolarizing, outward (K+), currents. The waveforms of action potentials in different regions of the heart are different reflecting to differences in the expression and/or the properties of the underlying ion channels. These differences contribute to the normal unidirectional propagation of excitation through the myocardium and to the generation of normal cardiac rhythms [4,5,6].

The cardiac electrical cycle has been divided in to five “phases”, four of them describing the AP contour and one the diastolic interval (Figures 1 and 2). Phase 0 refers to the autoregenerative depolarization, which occurs when the excitation threshold is exceeded. Phase 0 is supported by activation of two inward (depolarizing) currents, INa and ICaL. Membrane depolarization will quickly activate these channels and, with a delay of several milliseconds for INa and of tens of milliseconds for ICaL, inactivates them. Thus, membrane depolarization provides both the triggering and breaking mechanism for the autoregenerative depolarization. Although short-lived, INa is large and provides most of the charge influx required for AP propagation (see below). ICaL has a small component with fast activation/inactivation (ICaT) and a larger one with slower kinetics (ICaL). ICaL mediates most of Ca2+ influx required to trigger myocyte contraction and may support propagation under conditions in which INa is not expressed or functional (e.g. in the SA node). Phase 0 depolarization also activates K+ currents, which contribute to termination of this phase and to subsequent repolarization. Among these, the transient outward current (Ito) is fast enough to limit the depolarization rate during phase 0.

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Figure 2. Phases of a typical atrial and ventricular APs and underlying currents. The numbers refer to the five phases of the action potential. In each current profile, the horizontal line represents the zero current level; inward currents are below the line and outward currents are pointing upward.

Phase 1 is the initial phase of repolarization, mainly supported by Ito,f (the Ca independent Ito fast component –see section 3.3.1 for details), a potassium current that, similarly to INa, is activated and quickly inactivated by depolarization. Thus, Ito,f supports fast repolarization.

Phase 2, also named “plateau”, is the slow repolarization phase, which accounts for the peculiar configuration of the cardiac AP. The net transmembrane current flowing during phase 2 is small and it results from the algebraic summation of inward and outward components. The outward one (promoting repolarization) mainly consists of depolarization-activated K+ currents collectively named “delayed rectifiers” (IK). IK is actually the sum of rapid (IKr) and slow (IKs) components (and ultra-rapid in atria, IKur), carried by separate channels with different properties and pharmacology [7]. The inward phase 2 currents (opposing repolarization) are mostly carried by “window” components of INa and especially of ICaL, which flow when membrane potential (Vm) is such that the activated state of these channels is not yet completely offset by the inactivation process.

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This inward calcium movement is through long-lasting (L-type) calcium channels that open when the membrane potential depolarizes to about –40 mV. L-type calcium channels are the major calcium channels in cardiac and vascular smooth muscle. They are opened by membrane depolarization (they are voltage-operated) and remain open for a relatively long duration consequently causing the relative long plateau phase of AP (corresponding to the long ST segment of the ECG). The current generated by operation of the Na+/Ca2+ exchanger (INCX) can variably contribute to phase 2, according to the magnitude and course of the cytosolic Ca2+ transient and to the subsarcolemmal Na+ levels.

Phase 3 is the terminal phase of repolarization, which differs from phase 2 for its faster repolarization rate. Phase 3 is dominated by IKr and IK1, both characterized by a kinetic property, named “inward rectification” [8,9], suitable to support autoregenerative processes [10]. Initiation of phase 3 is probably supported by IKr, with a threshold determined by the balance between its onset and waning of phase 2 inward currents. IK1 takes over during the final part of phase 3 and effectively “clamps” membrane potential back to its diastolic value [11].

Phase 4 describes membrane potential during diastole. In myocytes expressing a robust IK1 (e.g. atrial and ventricular myocytes), Vm is stabilized at a value close to the current reversal and a significant current source is required to re-excite the cell. Under such conditions even small inward currents may cause progressive depolarization, eventually leading to re-excitation (automatic behaviour) [12]. Besides the time-dependent currents, specific for each AP phase, time-independent (or “background”) currents may also contribute to the whole AP course. These mainly include the Na+/K+ pump current (INaK) and the Na+/Ca2+ exchanger current (INCX). Direction and magnitude of these currents during the various AP phases will be determined essentially by their electromotive force. 3. Transmembrane ion channels in the heart 3.1. Voltage-gated Na+ (NaV) currents Voltage-gated cardiac Na+ (NaV) channels open extremely rapidly (within 3 ms) on membrane depolarization and in principle determine alone the rapidly rising phases of the action potentials recorded in mammalian ventricular and atrial myocytes and in cardiac Purkinje fibres [13, 14]. Although the properties of the NaV channels expressed in

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different cardiac cells are alike, the biophysical and pharmacological properties of these channels are quite different from NaV channels expressed in other excitable cells (neurons or skeletal muscle). Cardiac NaV channels, for example, are remarkably insensitive to the NaV channel toxin tetrodotoxin (TTX). On membrane depolarization, cardiac NaV channels activate and inactivate very rapidly [15]. Figure 3 show typical representative recordings from a single cell of INa in control and during superfusion with 10 and 30 μmol/L quinidine, a well-known sodium channel blocker Class IA antiarrhythmic drug [16].

Figure 3. The voltage gated fast sodium channel (INa). Panel A. Original INa trace recorded in rabbit ventricular myocytes (adapted from [16] with permission). The original recording show representative recordings of peak INa from a single cell in the absence (control) and the presence of 10 and 30 μM/L quinidine (a typical Class IA, i.e., a known sodium channel blocker, antiarrhythmic drug). Inset shows current–voltage curves in the absence (open circles) and the presence (solid circles) of 30 μM/L quinidine. Panel B. The schematic contribution of INa to the phase 0 (depolarization) on the action potential shape. Panel C. The biophysical double gating model of the sodium channel. Sodium channel has two gates, one activating gate (m) and one inactivating gate (h). In the resting (closed) state, the m-gates (activation gates) are closed, although the h-gates (inactivation gates) are open in order to wait activating pulse to open the channel. Rapid depolarization to threshold opens the m-gates (voltage activated), thereby opening the channel and enabling sodium to

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enter the cell. Shortly thereafter, as the cell begins to repolarize, the h-gates close and the channel becomes inactivated. Toward the end of repolarization, the m-gates again close and the h-gates open. This brings the channel back to its resting state. Summarizing, the channel can be activated only after reaching again the resting phase (i.e., the activating gate must first close and the inactivated gate must reopen). This phenomenon represents the subcellular basis of the refractoriness of the heart, i.e., the heart cannot be tetanised. The threshold for NaV channel activation is negative (approximately –55 mV), and the activation of these channels is strongly voltage dependent. Importantly, the inactivation is also voltage dependent, and so fast that cardiac NaV channels can undergo voltage-dependent inactivation without ever opening. At values corresponding to the action potential plateau in ventricular myocytes, present estimates are that 99% of the NaV channels are already in an inactivated, nonconducting state [17]. There is, therefore, a finite, albeit small (1%), probability of NaV channels being opened at potentials corresponding to the action potential plateau [17]. This slow component of NaV channel inactivation called late Na current (INaL) has indeed been described in normal human ventricular myocytes, [18]. The probability of NaV channel opening at depolarized potentials (i.e., during phase 2) is determined by the overlap of the curves describing the voltage dependences of channel activation and inactivation. Consistent with these predictions, electrophysiological studies reveal the presence of a sustained component of inward NaV current, i.e., a "persistent" NaV current, during prolonged membrane depolarizations, which was named “window” current. Although the "window" current is relatively small in comparison with the magnitude of the INa current, this current may contribute to action potential durations [19]. It has been reported that the expression level of the persistent NaV current component is different in various regions of the ventricles, differences that could contribute to the observed regional heterogeneities in ventricular action potential durations [20]. The impact of alterations in the "persistent" NaV channel window current on cardiac rhythms has now been unequivocally demonstrated with the identification of mutations in the gene, SCN5A, encoding the TTX-insensitive cardiac NaV channels in patients with an inherited form of long QT syndrome, LQT3 [21]. Several SCN5A mutations in different affected individuals/families have been identified and linked to Brugada syndrome and to conduction defects, in addition to LQT3 [22,23].

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3.2. Voltage-gated Ca2+ (CaV) currents In contrast to skeletal muscle, it has long been recognized that Ca2+ entry from the extracellular space is required for excitation-contraction coupling in the mammalian myocardium [2,24]. Several studies reported late that a "slow inward" (in comparison with NaV channel) current is carried by Ca2+ through a membrane conductance distinct from the Na+ movement [25]. Further studies characterized the time- and voltage-dependent properties of voltage-gated cardiac Ca2+ (CaV) currents in multicellular preparations, and later in isolated single cardiac cells as well [26,27]. Based on differences in the thresholds for channel activation two different types of CaV currents/channels were reported to be present in chick and rat sensory neurons [28,29]. These channels were termed high voltage-activated (HVA) and low voltage-activated (LVA) CaV channels, respectively. In the heart HVA and LVA CaV channels were first described in isolated canine atrial cells [30]. LVA CaV channels, also referred to as T-type Ca2+ channels [31], activate at relatively hyperpolarized membrane potentials, to about –50 mV, and these channels activate and inactivate rapidly [28,29]. On the contrary, HVA CaV channels (L-type), open on depolarization to more positive membrane potentials (approximately –20 mV), and inactivate over a longer time scale (several tens of milliseconds to seconds). Under physiological conditions, with Ca2+ as the charge carrier, HVA channels in most cells inactivate in <100 ms at depolarized voltages [30]. Figure 4 shows typical representative recordings from a dog ventricular single cell of ICaL in control and during superfusion with 10 μM/L dronedarone, an antiarrhythmic drug known to possess strong ICaL channel blocker effect [32].

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Figure 4. The voltage gated L-type calcium channel (ICaL). Panel A. Original ICaL trace recorded in dog ventricular myocytes (modified from [32] with permission). The original recording show representative recordings of ICaL from a single cell in the absence (control) and the presence of 10 μM/L dronedarone (known as iodine free “amiodarone” a typical multichannel antiarrhythmic drug, which is similar to amiodarone and a strong ICaL channel blocker). Panel B. The schematic contribution of ICaL to the phase 2 (depolarization) on the action potential shape. Lower Panels C. The biophysical double gating model of the calcium channel. Calcium channels, like sodium channel, have two gates, one activating gate (m) and one inactivating gate (h). In the resting phase (left panel) m gate is closed, while h gate is open waiting the activating pulse to open the channel. When membrane reaches the activating voltage threshold, m gate opens facilitating the entrance of the Ca2+ ion from the extracellular space into the cell (2nd panel). The resulting inward current is extremely fast activating (within 1-3 s, see panel A, left). The inactivating gate (h) fast closes (3rd panel) causing the relative fast inactivation (to about 50-150 ms) of the ICaL current. This medium fast inactivating calcium current is responsible for the relative long plateau phase of the AP shape. The double gated cardiac calcium channel has the intrinsic properties that it cannot be reactivated again from the

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inactive state to open the channel. The channel can be activated only after reaching the resting phase (4th right panel) (i.e., the activating m gate must first close and h the inactivated gate must reopen).

In the mammalian heart, L-type CaV channels currents appear to

be ubiquitously expressed. Apparently there are no interspecies and transregional differences in the properties and the densities of L-type CaV channel currents occur, suggesting similar molecular compositions of the underlying channel. Since the opening of cardiac L-type CaV channels in response to membrane depolarization is delayed relative to the NaV channels, ICaL contribute little to phase 0 depolarization in Purkinje, atrial and ventricular cells (Figures 1 and 2). The opening of L-type CaV channels and the Ca2+ entry through these channels is responsible for the action potential plateau (phase 2), which is particularly prominent in ventricular and Purkinje cells.

In addition, Ca2+ influx through the L-type CaV channels triggers Ca2+release from intracellular Ca2+stores and underlies excitation-contraction coupling in the working (ventricular) myocardium [1,2,33]. L-type CaV channels are also expressed in other heart regions as SAN and AVN cells, where they play an important role in action potential generation and automaticity regulation too [34]. Cardiac L-type CaV channels undergo rapid voltage- and Ca2+-dependent inactivation [35], processes that will also influence the action potential waveforms (Figure 2) by interfering the duration of the plateau (phase 2) and the time course of action potential repolarization. 3.3. Voltage gated K+ channels Voltage-gated K+ (KV) channels are the primary determinants of action potential repolarization in the mammalian myocardium. There is considerable electrophysiological and functional cardiac KV channel diversity as compared with cardiac NaV and CaV channels, there is considerable electrophysiological and functional cardiac KV channel diversity [36]. Based primarily on differences in time- and voltage-dependent properties and pharmacological sensitivities [6,36], two large classes of repolarizing cardiac KV currents have been distinguished: transient outward K+ currents (Ito) and delayed, outwardly rectifying K+ currents (IK). Ito currents activate and inactivate rapidly on membrane depolarizations to potentials positive to approximately –30 mV and underlie the early phase (phase 1) of repolarization (notch) in ventricular

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and atrial cells (Figure 2). The cardiac delayed rectifiers (IK) activate at relative similar membrane potentials, however having variable kinetics. IK currents determine the latter phase (phase 3) of repolarization back to the diastolic potential. Multiple types of myocardial Ito and IK channels were identified in various species. The distinct time- and voltage-dependent properties and differences in the densities and the biophysical properties of these channels contribute to variations in the waveforms of action potentials recorded in different cardiac cell types [4,36]. 3.3.1. Transient outward KV currents Although cardiac transient outward currents were first described in sheep Purkinje fibres and thought to reflect Cl– conductance [37], subsequent work demonstrated the presence of two transient outward currents with distinct properties and they were referred to as Ito1 and Ito2 [38]. Pharmacological studies revealed that the K+ selective Ito1 is blocked by high concentration of 4-aminopyridine (3-5 mM 4-AP) and is not affected by changes in extracellular Ca2+, whereas Ito2 cannot be blocked by 4-AP and is Ca2+ dependent [38].

Figure 5. The transient outward K+ channel (Itof). Panel A. Original Itof trace recorded in dog ventricular myocytes (This panel is modified from

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[42] with permission). Ito current was activated by 300 ms long depolarizing voltage pulses (inset top panel shows applied voltage pulse), while bottom panel shows the magnified Ito current recorded under the first 20 s long depolarizing pulse (corresponding with the magnification of Ito marked by the dashed line rectangle on the top panel). Panel B. The schematic contribution of Ito,f to the phase 1 (early repolarization, the notch phase of the AP) on the action potential shape. Panels C. The biophysical double gating model of the transient outward channel. Like fast sodium (see Fig 3) and inward calcium (see Fig 4) channels, Ito channels, have two gates, one activating gate (m) and one inactivating gate (h). In the resting phase (left panel) m gate is closed, while h gate is open waiting the activating pulse to open the channel. When membrane potential reaches the activating voltage threshold, m gate opens facilitating the exit of the K+ ion from the intracellular space to the extracellular space (2nd panel). The resulting outward current is extremely fast activating (within 1-3 s, see panel A, left). The inactivating gate (h) fast closes (3rd panel) causing the relative fast inactivation (to about 50-150 ms) of the Ito current. The double gated cardiac Ito channel has the intrinsic properties that it cannot be reactivated again from the inactive state. The channel can be activated only after reaching again the resting phase (4th panel) (i.e., the activating gate must first close and the inactivated gate must reopen).

In further studies, it was shown that the Ca2+-dependent Ito2 in Purkinje fibres and ventricular cells is a Cl– channel (Ca2+ activated chloride channel) [39]. The transient outward K+ currents, referred to variably by different laboratories as Ito, or Ito1 [40], have now been described in many cardiac cell types and in most species [41]. Comparison of the detailed biophysical properties of the transient outward K+ currents described in various cell types and species, however, suggested there are two types of transient outward K+ currents (Figure 5) [42]. This hypothesis is supported by several electrophysiological and pharmacological studies. In adult mouse and rat ventricular myocytes, for example, two transient K+ currents, termed Ito,fast (Ito,f) and Ito,slow (Ito,s), have been distinguished [43]. On membrane depolarization, mouse ventricular Ito,f channels activate and inactivate rapidly, and on membrane repolarization, these (Ito,f) channels recover rapidly from steady-state inactivation. Similar to Ito,f, mouse ventricular Ito,s channels activate and inactivate rapidly. Although the properties of the transient K+ currents in different cell types and species are similar and are amenable to classification as either Ito,f or Ito,s, there are large cell type and inter-species variations in the detailed biophysical

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properties of the (Ito,f and Ito,s) currents [41]. These observations suggest that there may well be subtle, albeit potentially important, cell type and/or species dependent molecular heterogeneity among Ito,f and Ito,s channels in different cell types and/or in different species. Although originally identified in Purkinje fibres, Ito,f is a prominent repolarizing current in atrial and ventricular myocytes in the other species including humans [44,45,46]. In humans and other larger mammals (as dog), Ito,f underlies the early phase (phase 1, notch) of repolarization in ventricular and atrial cells (Figure 5) and likely also contributes to determining the plateau (phase 2) [32,41,47,48]. Recent studies reported that the transient outward potassium current Ito,f, may influence not only indirectly by modulating the plateau duration, but also directly the phase 3 of the ventricular repolarisation in dog and human hearts [32,49]. However, there are exceptions. In guinea pig ventricular cells, for example, Ito,f has not been detected except when extracellular Ca2+ is removed [50]. These channels are absent in rabbit atrial and ventricular cells [46]. Nevertheless, there are transient KV currents in rabbit myocytes (typically referred to as transient inward current It), which inactivate slowly and recover from (steady-state) inactivation very slowly, whose properties more closely resemble mouse ventricular Ito,s than Ito,f and reflect possible different molecular structure background. Transient KV currents classified as Ito,f have also been shown to be expressed in (rabbit) SAN cells, although, similar to NaV currents, Ito,f densities vary markedly among SAN cells. In addition, when expressed, Ito,f appears to play a role in shaping action potential waveforms and in regulating automaticity in SAN cells [51]. Cells isolated from the (rabbit) AVN also express Ito,f, and detailed kinetic analysis of the currents reveals the presence of two components with distinct rates of inactivation and recovery [52]. It is unclear whether these findings reflect differences in the kinetic properties of a single type of Ito,f channel or if two distinct types of Ito channels are expressed in (rabbit) AVN cells. 3.3.2. Delayed rectifier KV currents

The delayed rectifier potassium current (IK) is a major outward current responsible for ventricular muscle action potential repolarisation [53]. This current was first described in 1969 by Noble and Tsien using the two-microelectrode voltage-clamp technique in sheep cardiac Purkinje fibre strands [54]. Since its discovery it has been examined in single isolated myocytes obtained from various regions of the heart in several mammalian species [7,55,56]. In most species, IK can be separated into

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rapid (IKr) and slow (IKs) components (Figure 6) that differ from one another in terms of their sensitivity to drugs, rectification characteristics, and kinetic properties [55,57,58,59].

Figure 6. The fast and the slow components of the delayed rectifier K+ channel (IKr and IKs). Upper panels. Original IKr (panel A) and IKs (panel B) traces recorded in human ventricular myocytes (modified from [58] and [59] with permission. IKr and IKs currents were activated using test pulses of 1000 ms (IKr) or 5000 ms (IKs) in duration to 20 mV from the holding potential of -40 mV. The decaying tail current at -40 mV after the test pulse were assessed as IKr or IKs tail currents. Panels C. The biophysical single gating model of the delayed rectifier potassium channels. IK channels unlike the previously presented INa, ICa and Ito channels (see Fig 3, 4, 5, respectively) have only one channel gate, which in resting state is closed (left panel). When cells are depolarized, the channel activates (2nd panel). In IKr channels this activation is fast, while in IKs channels is relatively slow. When cells are repolarized the channels is closed (3rd and 4th panels). IKs channels are not inactivating, thereby the closing mechanism is called deactivation. Previously it was thought that IKr behaves a similar way that IKs having one gate based activating and deactivating kinetics, until Spector et al [60] reported that IKr channels have in fact double gating mechanism like Ito channels. IKr channels

21

inactivate having extremely fast inactivation kinetics, however this inactivation display a voltage dependent behaviour. At positive membrane potential, the inactivation is blocked, while when membrane potential repolarizes and become more negative the inactivation blocking effect disappears and channels deactivate with a relative slow kinetics.

Although IKr activates rapidly, inactivates very rapidly and thereby

displays marked inward rectification, no inward rectification is evident for the slowly activating IKs [60]. In cardiac ventricular myocytes isolated from healthy human donor hearts, IKr activated fast and deactivated slowly and biexponentially (600 ms and 6700 ms) [61], while IKs exhibited slow and voltage independent activation at more positive than 0 mV (900 ms) and had fast and monoexponential deactivation kinetics [58,59]. This deactivation was voltage dependent, being fast at more negative (at -50 mV, 90 ms), and slow at more positive voltages (at 0 mV, 350 ms) [59]. In dog cardiac ventricular cells IKr activated fast and deactivated slowly and biexponentially, (360 ms and 3300 ms), while IKs activated slowly (800 ms) at voltage more positive than 0 mV, and deactivated rapidly and monoexponentially (150 ms) [55,62]. In the guinea pig, where it was first described, IKs activated very slowly, not saturating even after 5-7 s at +50 mV, and deactivated slowly, within 500-1000 ms [63]. Initially, it was not observed in the rabbit, but later studies revealed a large and consistent IKs in the rabbit ventricle [56; 64].

Considering the kinetic properties, the human cardiac slow delayed rectifier potassium current best resembles those measured in the dog (55; 62) and rabbit [56; 64] ventricle, but significantly differs from those found in the guinea pig heart [57]. IKs was augmented with an increase in sympathetic tone as a result of elevation in intracellular cAMP levels [58,65]. In canine [66] and human [67] atrial myocytes, a novel, very rapidly activating, and largely non-inactivating, outward KV current, now typically referred to as IKur [66], has been described. In most species, IKur is not detected in ventricular cells. It is likely that the expression and the properties of IKur, together with Ito,f, contribute to the more rapid repolarization evident in atrial, as compared to ventricular, myocytes (Figures 1 and 2). This suggests that IKur channels might represent a therapeutic target for the treatment of atrial arrhythmias without undesirable effects on impulse propagation, ventricular functioning, or cardiac output [68]. However, Wettwer et al. [69] reported that IKur

22

blockade may either prolong or shorten atrial APD depending on the disease status of the atria, which questioned the selective IKur blockade to atrial specific antiarrhythmic potential [69]. 3.3.3. Inward rectifier potassium currents

In addition to the depolarization-activated KV currents, inwardly rectifying K+ (Kir) currents, through IK1 channels, also contribute to myocardial action potential repolarization, particularly in ventricular cells [71]. As the "inward rectifier" terminology implies, Kir channels carry inward K+ currents rather than outward K+ currents [71]. Nevertheless, it is the outward K+ currents through these channels that are important physiologically because myocardial membrane potentials never reach values more negative than the K+ reversal potential (approximately –90 mV).

At the macroscopic level, IK1 channels have been characterized in human guinea pig and rabbit atrial and ventricular myocytes and in rabbit SAN cells [11,71,72]. The properties of the IK1 channels in each of these preparations are similar in that all are K+ selective, blocked by extracellular Ba2+ and intracellular Cs+ and strongly inwardly rectifying [72]. The strong inward rectification evident in cardiac IK1 channels is attributed to block by intracellular Mg2+, Ca2+ and polyamines [9,73,74]. The expression of IK1 is clearly reflected in the negative slope region (between approximately –50 and –10 mV) of the (total steady-state) myocyte conductance-voltage relation, which is prominent in ventricular myocytes, and smaller in atrial cells [72]. The fact that the strongly inwardly rectifying IK1 channels conduct at negative membrane potentials suggests that these channels play a primarily role in establishing the resting membrane potentials of Purkinje fibres, as well as of atrial and ventricular myocytes. Direct experimental support for this hypothesis was provided with the demonstration that ventricular membrane potentials are depolarized in the presence of large concentration of Ba2+ [70], which blocks IK1 channels. In addition, action potentials are prolonged, and phase 3 repolarization is slowed in the presence of extracellular Ba2+ (Figure 7), suggesting that IK1 channels also contribute to repolarization [75] and especially of repolarization reserve and arrhythmogenesis [58,77], particularly in the ventricular myocardium. Similar to the KV channels, IK1 densities and the detailed biophysical properties of the currents vary in different myocardial cell types. In the human heart, for example, IK1 density is more than two-fold higher in ventricular, than in

23

atrial, cells [77]. A recent study reported that ventricular IK1 current density in human is significantly lower than in dog; therefore, selective blockade of IK1 lengthens ventricular APD less in human than in dog [76]. However, this behaviour indirectly cause a more less pronounced IKr blockade induced APD lengthening in the dog than in human ventricle, therefore any IKr block drug effect tested in dogs can be underestimated when it is extrapolated to humans [76].

Figure 7. The inward rectifier potassium channel (IK1). Panel A. Original IK1 recording in a dog ventricular myocyte in the absence (control) and the presence of 10 μM/L BaCl2 (a typical IK1 channel inhibitor). Panel B. The effect of selective IK1 channel blockade by BaCl2 on the action potential recorded in dog right ventricular preparation (both two panels are adapted from [75] with permission). Panels C. The biophysical single gating model of the inward rectifier potassium channel. IK1 channels like IKs channels have only one channel gate, which is closed in the resting state. When cells are depolarized, the channel is activated, and later when the membrane is repolarized, the extremely slow deactivation of the channel occurs.

Another important and special ligand gated cardiac Kir channel

type is the acetylcholine sensitive potassium channel IK(ACh), which is

24

gated through a G protein-coupled mechanism mediated by muscarinic acetylcholine receptor activation (78). Physiologically, IK(ACh) channels are activated by the binding of G protein-subunits in response to the acetylcholine released on vagal stimulation (68). Although IK(ACh) channels are expressed in AVN, SAN, atrial, and Purkinje cells, and are activated by acetylcholine released on vagal stimulation, these channels are not thought to contribute appreciably to action potential repolarization under normal physiological conditions. Consistent with this hypothesis, targeted deletion of one of the Kir encoding IK(ACh) channels, Kir3.4, does not measurably affect resting heart rates. Interestingly, atrial fibrillation (AF) is not evident in Kir3.4 null mice exposed to the acetylcholine receptor agonist carbachol, suggesting that activation of IK(ACh) channels is involved in the cholinergic induction of atrial fibrillation (79). Similar to IKur, IK(ACh) is also an atrial specific channel. Thus, it is interesting whether selective IK(ACh) blockade cause repolarization lengthening effect, and be a basis for developing new antiarrhythmic drugs for treating chronic AF (cAF). In the study of Dobrev et al [80], IK(ACh) channels in cAF are constitutively active without any direct ligand stimulation. This observation suggests that selective IK(ACh) blockade may be a target for developing new antiarrhythmic drugs for treating cAF. Several pharmacological studies have supported this hypothesis [81]. 3.3.4. ATP sensitive potassium currents

The existence of K+ channels which could be blocked by internal

ATP in cardiac muscle was suggested a long time ago [82], but direct demonstration of the presence was possible only later with the aid of voltage-clamp techniques. The ATP sensitive potassium current (IKATP) channels, are inhibited by intracellular ATP, which are activated by nucleotide diphosphates; thus providing a link between cellular metabolism and membrane potential [83]. The current is time independent, and is activated when intracellular ATP concentration decreases below 1 mM. The reversal potential of IKATP (reported to be at -85 mV) is close to the K+ equilibrium potential [84], suggesting K+ as the major ion carrier. The current can be activated by several vasodilators such as cromakalim (Figure 8A), pinacidil (Figure 8B) and nicorandil, and is blocked by Ba2+, Cs+, tetraethylammonium, tedisamil (Figure 8A and B) [41,85,86]. However, under normal conditions the current is completely blocked by physiological intracellular ATP level. In the ventricular myocardium, the opening of IKATP channels is thought to be important under conditions of metabolic stress, as occurs during ischemia

25

or hypoxia, and to result in shortening action potential durations and minimizing K+ efflux [83].

Figure 8. The ATP sensitive potassium channel (IKATP). Panel A. Original IKATP recordings in a rabbit ventricular myocyte under control conditions, after opening of the IKATP channels with 50 µM cromakalim and after application of 1 µM tedisamil (an investigational antiarrhythmic drug having multichannel blocker profile) in the continuous presence of cromakalim. Panel B. The effect of 1 µM tedisamil on the pinacidil (10 µM) induced APD shortening in dog ventricular muscle fibres (all panels modified from [41] with permission).

Activation of IKATP contributes to the repolarization and shortening

of refractoriness in hypoxic/ischaemic conditions, which may cause life threatening cardiac arrhythmias. Drugs that inhibit IKATP can prevent or lessen the hypoxia/ischaemia induced shortening of repolarization and refractoriness and may protect from dangerous arrhythmias. On the other hand, however, growing evidence suggests that shortening of the APD can protect the myocardium from calcium overload, thus providing significant cardioprotection during acute ischaemia [87]. Although IKATP channels appear to be distributed uniformly in the RV and LV and through the thickness of the ventricular wall, these channels are expressed at much higher density than other sarcolemmal K+ channels, suggesting that action potentials could be shortened markedly when only very small numbers of IKATP channels are activated.

Table 2 summarizes the description of the ionic currents operating

in cardiac muscle.

26

Table 2. Summary of the ionic currents operating in cardiac muscle, their physiological roles and pharmacologic modulation.

Current Region Activated by Blocked by Main charge carrier

Effect

Fast inward sodium current (INa)

A, V, AVN, Purkinje

Depolarization beyond -60 mV

TTX, local anaesthetics

Na+ Rapid depolarization (phase 0), and maintenance of plateau (phase 2)

Inward calcium currents

L type ICa

A,V, SAN, AVN, Purkinje

Depolarization beyond -30 mV

Cd2+, Mn2+, organic Ca channel blockers

Ca2+ Depolarization, maintenance of plateau (phase 2), calcium induced calcium release

T type ICa

A, SAN, AVN

Depolarization beyond -60 mV

mibefradil, amiloride, flunarizine

Ca2+ Depolarization, automaticity

Transient outward currents

Ito,f A, V, SAN, AVN, Purkinje

Depolarization beyond -20 mV

4 aminopyridine (4-AP), heteropoda toxin

K+ Rapid repolarization (phase 1)

Ito,s A, V Depolarization and intra-cellular Ca2+

Intracellular EGTA, anion channel blockers (SITS, DIDS)

Cl- Early repolarization (notch, phase 1)

Delayed rectifier potassium currents

IKr A,V, SAN, AVN, Purkinje

Depolarization beyond -20 mV and IKr activators (NS3623, NS1643)

Class IA and III antiarrhythmic drugs (quinidine, dofetilide, E-4031, d-sotalol)

K+ Termination of repolarization (phase 3)

IKs A,V, SAN, AVN,

Depolarization beyond 0 mV and

L-735,821, HMR-1556, Chromanol

K+ Termination of repolarization (phase 3)

27

Purkinje sympathetic activation

293B

IKur A Depolarization beyond -30 mV

Low concentration (50 µM) 4-AP, AVE0118

K+ Modulation of plateau (phase 2) and termination of repolarization (phase 3)

Inward rectifier potassium currents

IK1 A,V, AVN, Purkinje

Depolarization Ba2+, indapamide

K+ Reduction of membrane conductance during plateau (phase 2), termination of repolarization (phase 3) and restoring of the membrane resting potential (phase 4)

IKACh A, SAN, AVN

Depolarization and stimulation of muscarinic receptor (carbachol)

Tertiapine, Ba2+

K+ Shortening of repolarization, decreases resting membrane potential (phase 4)

IKp A, V, undefined in other regions

depolarization unknown K+ Decreases membrane potential

ATP sensitive potassium current

IKATP A,V Depolarization and decrease of intracellular ATP, vasodilator agents (cromakalim, pinacidil, nicorandil)

glibenclamide K+ Shortening of repolarization during hypoxia

Abbreviations: A - atria; V – ventricle; SAN – sinoatrial node; AVN – atrioventricular node; SITS (4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid); DIDS (4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt); E-4031 (IUPAC name: (1-

28 [2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonyl-aminobenzoyl)piperidine); L-735821 (IUPAC name: (E)-3-(2,4-dichlorophenyl)-N-[(3R)-1-methyl-2-oxo-5-phenyl-3H-1,4-benzodiazepin-3-yl]prop-2-enamide); HMR-1556 (IUPAC name: N-[(3R,4S)-3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methylethanesulfonamide); chromanol-293B (IUPAC name: trans-N-[6-Cyano-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-4-yl]-N-methyl-ethanesulfonamide); AVE0118 (IUPAC name: 2’-{[2-(4-methoxy-phenyl)-acetylamino]-methyl}-biphenyl-2carboxylic acid (2-pyridin-3-yl-ethyl)-amide 3.3.5. Background potassium channels

A small and time-independent potassium conductance has been described in guinea-pig cardiac myocytes [88]. None of the cloned 6Tm–1P or 2Tm–1P channels encode such current, but several recently cloned tandem pore subunits (4Tm–2P) encode currents with this ‘leak’ current behaviour, e.g. the fairly ubiquitous TWIK subunit (“Tandem of P-domains in a Weakly Inward rectifying K+ channel”) [89]. Presence of TWIK mRNA was found in large amount in human atrial and ventricular preparations [90]; however, the properties of the current formed by these channels are still poorly understood. A related subunit TASK (“TWIK-related acid sensitive K+ channel”) is also highly expressed in the heart [91]. The channel is sensitive to pH variations in the physiological range, and contains a C-terminal PDZ binding motif. This relatively new family is rapidly expanding and subunits responsible for the cardiac background potassium channel (IKp) component yet to be established. 4. Concluding remarks This chapter has focused on ion channels that contribute to AP formation in normal hearts in physiological conditions. However, several ion channels play only a role in pathophysiological conditions related to different cardiac diseases or during early development. Several studies have indicated that ion channels function properly only in the presence of various exo- and/or endogenous regulatory molecules; moreover, next to several identified gene mutations and polymorphisms, ion channel expression are strongly influenced by molecular variants (genes and/or small noncoding RNAs and microRNAs). The investigations of these effects on channel expression

29

near electrophysiological properties of the transmembrane ionic currents are of importance, because: i) exploring their function may provide novel mechanistic insights into the pathophysiology of several specific cardiac diseases; ii) the investigation of the transmembrane ion current at organ, cellular and subcellular levels may identify new and more specific targets to treat arrhythmias, which will provide a more potent effect without proarrhythmic side effects that are present with the currently used antiarrhythmic drugs. Acknowledgements This work was supported by a grant of the Ministry of National Education, CNCS – UEFISCDI, project number PN-II-ID-PCE-2012-4-0512 and grants by the Hungarian Scientific Research Fund (OTKA NN109904 and ANN113273), by the Hungarian Academy of Sciences and by TÁMOP 4.2.4. A/2-11-1-2012-0001 „National Excellence Program – Elaborating and operating an inland student and researcher personal support system” key project to Norbert Jost. The project was subsidized by the European Union and co-financed by the European Social Fund.

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