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European Heart Journal Supplements (2003) 5 (Supplement G), G19—G25 If inhibition: a novel mechanism of action D. DiFrancesco Department of General Physiology and Biochemistry, Laboratory of Molecular Physiology and Neurobiology, Milan, Italy KEYWORDS Cardiac rate; Funny current; If blockers; Pacemaker; Sinoatrial node Aims It is well established that the cardiac pacemaker (‘funny’, or If) current plays an important role in the generation and autonomic modulation of cardiac rate by controlling the rate of diastolic depolarization. Here, the properties of If and the criteria that permit identification of If activation as the main mechanism responsible for diastolic depolarization are briefly summarized. The relationship between If inhibition by specific If channel blockers (rate-reducing agents) and reduction in pacemaker rate is also described. Methods and results The If data reported here were collected from rabbit sinoatrial node cells that were isolated and patch-clamped. Cs+ ions and, more efficiently, ‘ratereducing’ agents block If and reduce the steepness of diastolic depolarization and frequency in spontaneously active sinoatrial node myocytes. Ivabradine (Procoralan®; Servier, Neuilly-sur-Seine, France), a recently developed molecule, blocks If channels when they are open and preferentially when the current is outward. Conclusions If controls the slope of diastolic depolarization and cardiac frequency, and its inhibition causes heart rate reduction. The current-dependent blockade of If with ivabradine leads to a specific and use-dependent, heart rate reducing effect that may have therapeutic applications in clinical settings. © 2003 The European Society of Cardiology. Published by Elsevier Science Ltd. All rights reserved Introduction The ability of sinoatrial node pacemaker cells to pace spontaneously depends on the presence in these cells of the diastolic depolarization phase of the action potential (phase 4). At the end of an action potential, the repolarization process terminates at a maximum diastolic depolarization (MDP) level of around —60 to —70 mV; this level of depolarization is greater than those that are typical of working cardiac muscle (i.e. under —80 mV). The membrane voltage does not set to rest, but slowly Correspondence: Dario DiFrancesco, Department of General Physiology and Biochemistry, Laboratory of Molecular Physiology and Neurobiology, via Celoria 26, 20133 Milano, Italy. depolarizes until it arrives at the threshold for activation of the faster transient, which leads to another action potential, thus producing rhythmic activity (Fig. 1). In the mammalian heart, diastolic depolarization is typically found in cardiac regions that are able to pace spontaneously and is absent in the working myocardium, except under specific pathological conditions. The diastolic depolarization, or pacemaker, phase of the action potential serves two main purposes: first, it is responsible for generation of spontaneous activity of the sinoatrial node, and hence of cardiac rhythm; and second, it mediates regulation of pacemaker frequency by the autonomic nervous system. Therefore, acquiring a better understanding of the ionic and cellular mechanisms that underlie generation of pacemaker depolarization, and more 01520-765X/03/0G0019 + 07 $35.00/0 © 2003 The European Society of Cardiology, Published by Elsevier Science Ltd. All rights reserved. G20 D. DiFrancesco (a) Iso 0.3 µM control control ACh 0.03 µM 100 ms 100 ms 50 mV 50 mV (b) —35 100 ms —35 100 ms control 500 pA 250 pA —85 Iso 1 µM (c) Iso 1 µM control mV —100 —50 ACh 0.3 µM —85 control 1.0 0.5 0 control ACh 1 µM mV —100 —50 1.0 0.5 0 Fig. 1 If mediates chronotropic modulation of pacemaker activity by the autonomic transmitters. (a) Spontaneous action potentials recorded in two isolated sinoatrial node myocytes in control conditions and during perfusion with isoprenaline (Iso) 0.03 µmol/l (left) or acethylcholine (ACh) 0.03 µmol/l (right), as indicated. Note acceleration by Iso and slowing by ACh. (b) If records obtained during hyperpolarization to —85 mV from a holding potential of —35 mV in two different cells in control Tyrode solution and during perfusion with Iso 1 µmol/l (left) or ACh 0.3 µmol/l (right). Note that If increased with Iso and decreased with ACh. (c) The If activation curve shifted by about 13.0 mV in the presence of Iso 1 µmol/l (left), and in a different cell by about —10.6 mV in the presence of ACh 1 µmol/l (right). Activation curves were measured by a voltage ramp method on the left13 and by a voltage step activation protocol on the right.2 All records were taken at a temperature of 35°C. generally of pacemaker activity, remains a central issue in cardiac physiology. Because rhythmic firing is a basic type of activity of other cell types, typically of neurones, this issue is in fact central to physiology as a whole. What is known of the mechanisms that underlie pacemaker generation? The MDP is depolarized in sinoatrial node cells; furthermore, at the basis of repetitive activity is a slowly progressing depolarization. Those observations indicate that an inward current component must be expressed specifically in these cells and be activated during the slow depolarizing phase. An inward current that is activated on hyperpolarization in the diastolic range of voltages was indeed described during the late 1970s, and was shown to have properties sufficient for generating phase 4 of the pacemaker cell action potential.1 The ‘funny’ (If) current is so termed because of its unusual features, including that of being an inward current that is activated on hyperpolarization and not on depolarization like other known currents. If was found to be able not only to generate a slow depolarization but also to control its slope, and hence the rate of cardiac rhythm, under the influence of adrenaline (epinephrine); in other words, If was shown to mediate the positive chronotropic action of beta-receptor stimulation by sympathetic stimuli.2 Early studies of pacemaker activity in the conduction tissue (Purkinje fibres) had already led to the identification of a ‘pacemaker’ current. However, this had been erroneously interpreted as a pure K+ current (IK2), outward and deactivating on hyperpolarization in the pacemaker range of voltages.3 This inaccurate description held until the early 1980s, when the discovery of If in the sinoatrial node raised serious reservations as to the possibility that two pacemaker currents of different ionic nature could exist. This issue was resolved in 1981 with the discovery that the Purkinje fibre’s IK2 had been wrongly interpreted and was indeed identical to the nodal If.4,5 Those findings revealed the existence of a general mechanism for inducing spontaneous activity in cardiac cells. Since its discovery, the If current has been the object of several studies aimed at understanding its ionic, kinetic and modulatory properties.6,7 If-like currents were also described in neurones and shown to be important to several aspects of the regulation of neuronal excitability.8 Identification of the molecular subunits contributing to form the native pacemaker channels subsequently provided new tools with which to investigate their molecular properties. The newly cloned family of hyperpolarization-activated, cyclic-nucleotidegated (HCN) channels comprises four isoforms that are variously distributed among cardiac cells and neurones.9—11 Here, the properties of native cardiac pacemaker channels are briefly summarized, and some basic aspects of channel blockade by specific If inhibitors (rate-reducing agents) are discussed. Methods The experiments reported here were conducted in sinoatrial node cells isolated from rabbit hearts and patch-clamped in the whole-cell configuration. The methods for isolation and storage of single cells from the central sinoatrial If inhibition: a novel mechanism of action node area were reported in detail previously.12 The cells studied were perfused with normal Tyrode solution under the stage of an inverted microscope and were patch-clamped with pipettes filled with an intracellular-like solution. Control and test solutions were delivered via a fastperfusion pipette located on top of the cell under study. The compositions of the perfusing and pipette solutions are described elsewhere.13 Temperature was controlled in all experiments and was set to between 32 and 35°C. Data analysis was performed off-line. Results and discussion The If current and pacemaker activity The questions as to whether If is indeed responsible for generation of diastolic depolarization and the extent to which it participates in the control of cardiac rate have been subject to intense debate since the current was first described in the sinoatrial node.7,14 An indication of the possible functions of If can easily be derived from its elementary properties.7,12 If is a mixed Na+ and K+ current that is activated on hyperpolarization to voltages within the pacemaker range. Its reversal potential in normal Tyrode solution is approximately —10 to —20 mV, and the current is therefore inward at diastolic voltages. Its activation threshold is about —40 to —50 mV, and the current is fully activated at about —100 to —110 mV. If activates slowly upon hyperpolarization, with a time constant that becomes shorter at more negative voltages (about 1 s at —55 mV and 0.5 s at —75 mV), and it deactivates rapidly upon depolarization to voltages within the plateau range (about 30 ms at 15 mV).15 Based on these features, it is possible to understand how If works. Because the activation range of If overlaps that of diastolic depolarization (Fig. 1a and c), repolarization of the action potential will lead to If activation; and because If is inward at diastolic voltages, its activation will lead to generation of a slowly developing depolarization process — the diastolic depolarization. Together with its elementary properties, several other experimental findings reported since the discovery of If contribute to a wealth of evidence highlighting the fundamental role of this current in the spontaneous activity of cardiac pacemaker cells, as well as in the control of various aspects of cell excitability, including spontaneous firing of neurones.7,8 Among those findings, especially relevant is the modulation of cardiac rate by G21 autonomic transmitters, in which the basic role of If has been demonstrated, and the evidence that pharmacological blockade of If channels with specific blocking molecules affects the rate of diastolic depolarization, and hence cardiac rate, in a rather specific manner. These aspects are discussed below. Heart rate is controlled by If The sinoatrial node, or pacemaker, region of the mammalian heart is the most densely innervated by both adrenergic and cholinergic branches of the autonomic nervous system. Through innervation of the sinoatrial node, the autonomic nervous system exerts control over the chronotropic state of the heart; sympathetic stimulation accelerates and parasympathetic stimulation slows cardiac frequency by acting through beta-adrenergic and muscarinic receptors, respectively. What are the cellular mechanisms that are responsible for the autonomic modulation of rate? Low sympathetic and parasympathetic agonist concentrations modify the rate via changes in the steepness of diastolic depolarization. In Fig. 1a, the effects of beta-adrenergic (left) and muscarinic stimulation (right) on spontaneous activity recorded from isolated sinoatrial node cells are shown. It is apparent that the rate acceleration induced by 0.3 µmol/l isoprenaline involves a steeper slope of the diastolic depolarization, with little or no change in other action potential properties; similarly, a shallower slope of the diastolic depolarization is the only determinant of the rate slowing induced by 0.03 µmol/l acetylcholine (ACh). The fact that neurotransmitter-induced rate changes are concentrated on the pacemaker depolarization implies that the mechanisms that operate at low agonist concentrations are those that specifically regulate phase 4 of the action potential. This notion is in keeping with the observation, originally reported in 1979,1 that If is increased by adrenaline in a manner that is compatible with adrenaline-induced rate acceleration. The stimulatory action of betareceptor activation on If recorded in a sinoatrial node is shown in Fig. 1b (left). Following this initial finding, further investigation demonstrated that beta-adrenergic stimulation increases If via a shift in the current activation curve to more positive voltages.12,13 An example of this effect is shown in Fig. 1c (left). Clearly, during betareceptor stimulation a larger fraction of If current is available at each voltage, which explains the acceleration in rate of diastolic depolarization. G22 The rightward shift in the If activation curve is caused by an increase in the level of intracellular cyclic adenosine monophosphate (cAMP) — the second messenger that mediates If modulation.7 The increased rate of spontaneous activity associated with moderate sympathetic activity can therefore be explained by the following cascade of cellular events: beta-receptor stimulation activates adenylate cyclase and increases the synthesis of cAMP, which binds to If channels and shifts their activation curve to more positive voltages, eventually leading to an increased inward current and a steeper slope of diastolic depolarization. An opposite series of events regulates the rate slowing caused by low concentrations of ACh.16 In contrast to the hypothesis, long held by cardiac physiologists, that the negative chronotropic action of ACh was exclusively due to activation of an ACh-dependent K+ current (IK,ACh), comparative experiments in sinoatrial node cells demonstrated the following:17 that low doses of ACh inhibit If, and slow the rate, without affecting IK,ACh; and that an increase in K+ conductance is only obtained with much higher ACh concentrations. The decrease in If involves reduced cAMP production and a shift of the current activation curve to more negative voltages.18 Figure 1b and c (right) shows samples of the ACh-dependent decrease in If during hyperpolarizing steps and of the negative shift of the If activation curve. The ACh-mediated If inhibition has special physiological relevance because it is responsible for the vagal regulation of basal cardiac rhythm; additionally, as discussed below, this process is the physiological equivalent of pharmacological interventions aimed at reducing heart rate by inhibition of the If current. Heart rate reduction induced by If blockers As mentioned above, the question of the relevance of If to pacemaker depolarization has long been discussed7,14 and has been addressed by specific experimental protocols aimed at determining the effect on rate of If changes (for example, see DiFrancesco).19 Clearly, the evidence discussed above that modulation of If by autonomic transmitters leads to pacemaker rate modulation highlights the essential role of If. More direct evidence of If involvement can be gained from the use of agents that specifically interact with If channels, such as If channel blockers, by which a ‘pharmacological dissection’ of the contribution of If to rate can in theory be estimated. It is essential for complete D. DiFrancesco dissection that a highly specific If channel blockade is induced. Figure 2a shows the action of Cs+, a known blocker of If, on the spontaneous action potentials and on the fully activated I/V relation of If, as measured in sinoatrial node cells. The If block is voltage dependent and increases at more negative voltages, causing a distinct outward rectifying behaviour and a region of negative slope at hyperpolarized voltages (Fig. 2a, bottom). These features were incorporated into a model of If channel block by Cs+, according to which external Cs+ ions must enter the channel for a fraction (about 70%) of the membrane electrical field before binding to the blocking site.20 The voltage dependence of If block thus reflects the voltage dependence of Cs+ access to its binding site within the channel pore. If current block occurs rapidly upon hyperpolarization to negative voltages and is as rapidly relieved on depolarization to positive voltages, reflecting the fact that Cs+ ions are ‘pushed’ toward, or ‘pulled’ from, the binding site, according to the voltage applied. As shown in the top panel of Fig. 2a, spontaneous activity is slowed but not halted by Cs+ — an observation often used to refute the relevance of If to pacemaker generation.22 However, it is apparent from the I/V curve modification in the presence of Cs+ (bottom panel) that even millimolar Cs+ concentrations do not fully block If at diastolic voltages (23—38% in the range —65 to —45 mV).7 Note also that Cs+ slightly prolongs the action potential duration, possibly reflecting a reduction in the delayed K+ conductance; this is not unexpected because Cs+ is a known K+-channel blocker.23 Quite a different type of block is observed with UL-FS49 (zatebradine) — a ‘rate-reducing’ agent (Fig. 2b). If block by zatebradine is also voltage dependent, but it has a voltage dependence that is opposite to that of Cs+ block (i.e. it increases at more positive potentials; bottom panel of Fig. 2b). Analysis of the action of zatebradine21 indicates that If block is exerted from the intracellular side of the membrane, and that it occurs only when channels are in the open state (‘open channel blocker’). The voltage dependence of block can be reproduced by assuming that zatebradine molecules enter the pore from the intracellular side for a fraction of the membrane electrical field before binding to the blocking site.21 When measured from the external membrane side, this fraction is about 61%; this value is not too dissimilar from that of Cs+ block, suggesting that both agents may interfere with the ion flow through If channels at similar positions in the pore. If inhibition: a novel mechanism of action (a) (b) 200 ms 50 mV G23 200 ms 50 mV zatebradine 0.3 µM Cs 2 mM 200 mV —100 Cs 5 mM —50 mV —100 —50 pA zatebradine 3 µM —200 pA —1000 —400 Fig. 2 If blockers reduce the rate of diastolic depolarization. (a) Cs+ (5 mmol/l) caused a voltage-dependent block of the fully activated I/V relation of If, with the fractional block increasing steeply with hyperpolarization (bottom). If block by Cs+ (2 mmol/l) slowed pacemaker activity in another cell by diminishing the slope of diastolic depolarization (top); however, a modest prolongation of action potential duration reflected partial block of K+ currents. (b) Zatebradine (3 µmol/l) — a rate-reducing agent — also blocked the fully activated If in a voltage-dependent manner, but with fractional block decreasing with hyperpolarization (bottom). Low concentrations of zatebradine (0·3 µmol/l) were able to decrease spontaneous rate by modifying only the diastolic depolarization (top), indicating a more specific If blocking action than with Cs+. Fully-activated I/V relations in (a) and in the control curve in (b) were measured as previously published (see, for example, DiFrancesco).20 The I/V curve in the presence of zatebradine in (b) was calculated as previously detailed.21 Temperature was 35°C throughout. As shown in the top panel of Fig. 2b, the rate slowing induced by zatebradine 0.3 µmol/l is due to a reduction in diastolic depolarization slope, without changes in duration of action potential. An effect exerted solely on phase 4 of the action potential, although variable in degree of specificity, is typical of rate-reducing agents (see below) and confirms the existence of a direct relationship between If inhibition and reduction in spontaneous cardiac rate; this is in agreement with the view that If activation during diastolic depolarization is indeed the mechanism responsible for initiation and control of heart beat. Action of the rate-reducing agent ivabradine A recently developed substance, namely Procoralan® (ivabradine; Servier, Neuilly-surSeine, France), exhibits a high degree of specificity as an If-blocking molecule,24 and exerts a correspondingly specific action as a ratereducing agent (i.e. lack of inotropic side effects when tested in vivo,25,26 suggesting potentially useful applications in clinical settings. Ivabradine acts on If channels more selectively than does Cs+ and zatebradine; the half-block concentration of ivabradine measured upon repetitive activation/ deactivation steps is 1.5 µmol/l,27 whereas that of zatebradine is 80 µmol/l,28 and 5 mmol/l Cs+ blocks less than 38% of the current at voltages in the range —65 to —45 mV.7 Detailed investigation of the If-blocking properties of ivabradine shows that the drug blocks from the inside24 and requires open channels to access its blocking site, acting therefore as an open channel blocker. This property is illustrated in Fig. 3. In Fig. 3a, ivabradine (3 µmol/l) was superfused during an activation/deactivation protocol, in which steps to —100/+5 mV were repetitively applied every 6 s from a holding potential of —35 mV; the If amplitude measured at —100 mV underwent a slow, time-dependent inhibition caused by channel blockade until after about 180 s, when it settled to a steady-state level corresponding to approximately 75% reduction (compare traces in control and after full blockade in the upper panel). Note that the blocking action was reversible and that the current recovered toward its original amplitude following washout. If, on the other hand, ivabradine was perfused while holding the membrane at —35 mV (when If channels are closed) for 100 s, no blockade developed; only by reapplying the normal activation/deactivation protocol was development of blockade restored (Fig. 3b). This proves that ivabradine can access its binding site and exert its blocking action only when If channels are open. Similar experiments show that not only block onset but also removal of block requires open channels (not shown). Thus, the action of ivabradine is compatible with the idea that drug molecules can be ‘trapped’, when bound, by the channel voltage-dependent gates located at the intracellular entrance of the pore. A similar result was obtained with another rate-reducing agent, namely ZD 7288, in studies performed in heterologously expressed HCN channels.29 The steady-state blockade of If by ivabradine, as measured in the fully activated I/V relation, has some peculiar properties when compared with other rate-reducing agents. In Fig. 4, the fully activated I/V relation measured in a single cell in control conditions (closed circles) and that obtained by multiplying the control I/V curve by the fractional steady-state block curve at 3 µmol/l ivabradine (open circles) are shown. Clearly, substantial block is exerted only at voltages in which If is outward, whereas the inward part of the I/V relation is little affected. These data G24 D. DiFrancesco 2s (a) 250 pA 0 pA —100 1 s 0 * 500 0 pA/pF 1 2 3 s 0 —1000 1 1 ivabradine 3 µM 500 mV 3 —100 2 indicate that ivabradine blocks If channels preferentially when the current flow across channels is outward, whereas a very modest inhibition occurs when the current is inward. The block by ivabradine cannot therefore be simply explained in terms of a voltage-dependent block such as is proposed for zatebradine,21 or in terms of a channel state-dependent block as proposed for ZD 7288.29 It is instead consistent with the idea that ivabradine competes with permeating ions at one of their binding sites along the permeating pathway in the channel pore. Conclusion The data discussed above show that If activation is the process that is mainly responsible for generation of diastolic depolarization. Control of the diastolic depolarization rate, and hence of cardiac chronotropy, by the autonomic nervous system is mediated by changes in the degree of activation of If via changes in cAMP levels. If If activation is a determinant of diastolic depolarization and of the frequency of spontaneous activity, then modifications in If should lead to parallel changes in heart rate. Indeed the finding that ‘heart rate reducing’ agents — molecules that induce heart rate reduction without inotropic side effects — act by specific If blockade confirms the essential role of If in pacemaking. —50 0 50 —10 * ivabradine 3 µM Fig. 3 Ivabradine is an open channel blocker. Activation/deactivation voltage step protocols (—100 mV/+5 mV) were applied at a frequency of 1/6 Hz and the current amplitude was measured during hyperpolarization to —100 mV. (a) Time course of If during perfusion with ivabradine 3 µmol/l, showing the timedependent current inhibition by the drug (bottom). Sample traces in (1) control and (2) after steady-state block are plotted in the top panel. (b) In a different cell, the same protocol was interrupted and the cell was held at —35 mV while perfusing ivabradine (3 µmol/l); the activation/deactivation protocol was resumed after 100 s. Traces in (1) control, (2) just after the 100 s period at —35 mV and (3) after steady-state block are shown in the top panel. Temperature was 32.5°C. control 10 * pA 2 —200 —300 2 20 2s (b) 500 pA —20 Fig. 4 Voltage dependence of If block by ivabradine 3 µmol/l. The fully activated I/V relation of If was obtained in a sinoatrial node myocyte (filled circles) as previously described (see DiFrancesco et al.12) and fitted to a second order polynomial curve (full line). Fitted values were multiplied by mean fractional block values previously measured (not shown) at appropriate voltages to yield a fully activated I/V relation, corresponding to steady-state block by ivabradine (open circles, line through points). Procoralan (ivabradine), a recently developed rate-reducing substance, inhibits If channels with atypical blocking features, leading to a specific use dependence. The selectivity (the interaction with the If channel vs other channels) and specificity (the current dependence vs voltage dependence of If inhibition) characteristics of Procoralan appear therefore to render that agent potentially safe and effective in various pathologies that require independent reduction in heart rate. References 1. Brown HF, DiFrancesco D, Noble SJ. How does adrenaline accelerate the heart? Nature 1979;280:235—6. 2. DiFrancesco D, Tromba C. Inhibition of the hyperpolarization-activated current (If) induced by acetylcholine in rabbit sino-atrial node myocytes. 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