Physiological and Pharmacological Insights into the ...

Cardiovascular & Haematological Disorders-Drug Targets, 2006, 6, 000-000

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Physiological and Pharmacological Insights into the Role of Ionic Channels in Cardiac Pacemaker Activity B. Couette, L. Marger, J. Nargeot and M. E. Mangoni* CNRS UMR5203, Montpellier, F-34094 France; INSERM, U661, Montpellier, F-34094 France; Université de Montpellier I, Montpellier, F-34094 France; Université de Montpellier II, Montpellier, F-34094 France; Institut de Génomique Fonctionnelle, Département de Physiologie Abstract: The generation of cardiac pacemaker activity is a complex phenomenon which requires the coordinated activity of different membrane ionic channels, as well as intracellular signalling factors including Ca2+ and second messengers. The precise mechanism initiating automaticity in primary pacemaker cells is still matter of debate and certain aspects of how channels cooperate in the regulation of pacemaking by the autonomic nervous system have not been entirely elucidated. Research in the physiopathology of cardiac automaticity has also gained a considerable interest in the domain of cardiovascular pharmacology, since accumulating clinical and epidemiological evidence indicate a link between an increase in heart rate and the risk of cardiac mortality and morbidity. Lowering the heart rate by specific bradycardic agents in patients with heart disease constitutes a promising way to increase cardioprotection and improve survival. Thus, the elucidation of the mechanisms underlying the generation of pacemaker activity is necessary for the development of new therapeutic molecules for controlling the heart rate. Recent work on genetically modified mouse models provided new and intriguing evidence linking the activity of ionic channels genes to the generation and regulation of pacemaking. Importantly, results obtained on genetically engineered mouse strains have demonstrated that some channels are specifically involved in the generation of cardiac automaticity and conduction, but have no functional impact on the contractile activity of the heart. In this article, we will outline the current knowledge on the role of ionic channels in cardiac pacemaker activity and suggest new potential pharmacological targets for controlling the heart rate without concomitant negative inotropism.

Key Words: Pacemaker activity, sinus node, Ionic channels, Ca2+ channels, HCN channels, K+ channels, channels expression pattern, genetically-modified mice, cardioprotection. INTRODUCTION By commencing the contraction of the working myocardium, the spontaneous pacemaker activity of the heart ensures the correct time setting of the blood flow into vessels, thereby playing a key role in integration of vital functions of the organism. The automaticity of the heartbeat has fascinated scientists, philosophers and even artists well before the establishment of the foundations of the modern science. The persistency of the heart beating outside the body is in fact, a naturally intriguing and fascinating observation. In the medical practice, the presence or absence of the heartbeat has established the boundary between life and death for centuries. Even at the present days, the link between the heartbeat and our intimate concept of life is powerful, and still constitutes one important issue in the setting of standards for the clinical and ethical definition of death [1, 2]. Beside the doubtless fascination exerted by the heartbeat, the study of the mechanisms underlying cardiac pacemaker activity is a challenging and demanding research field in the modern Physiology. The ionic basis of cardiac pacemaking have been investigated since the end of the sixties by applying the double-electrode voltage-clamp technique on strips of spontaneously active tissue coming from the Purkinje fibers *Address correspondence to this author at the Institut de Génomique Fonctionnelle (IGF), CNRS UPR5203, 141, rue de la Cardonille, F-34094 Montpellier Cedex 5, France; Phone: (33) 499 61 99 39; Fax: (33) 499 61 99 01; E-mail: [email protected] 1871-529X/06 $50.00+.00

network [3-7] and the sino-atrial node (SAN) [8, 9]. During this pioneering research period, the principles of functioning of the pacemaker action potential and some of ionic currents potentially involved in the generation of automaticity have been described. During the last twenty years, the use of the patch-clamp technique on isolated pacemaker cells has allowed the description of different families of ionic channels, as well as the identification of the intracellular signalling pathways involved in the autonomic regulation of ionic channels activity [10, 11]. Also, the application of Ca2+ imaging to spontaneously beating cells has revealed a new potential role for intracellular Ca2+ release in the regulation of pacemaker activity. The growing knowledge on the cardiac pacemaker mechanism is reflected by the increasing complexity of numerical models of automaticity that have been developed from the now classical model of automaticity of Purkinje fibers by DiFrancesco & Noble [12], to the recently developed models of SAN pacemaking In these models, specific SAN properties such the heterogeneity in the expression of ionic channels [13] or the regulation of automaticity by intracellular Ca2+ release [14, 15] have been included. Numerical models of pacemaker activity are now beginning to play an important role in the development and testing of new drugs, and constitute a key part into the development of the cardiac Physiome project [16]. Research on cardiac pacemaking has now entered its “postgenomic” phase. Indeed, the molecular cloning of cardiac ionic channels and the use of new genetically modified © 2006 Bentham Science Publishers Ltd.

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animal models such as the mouse or zebrafish (Danio rerio) has yielded new insights into the functional role of ionic channels in the generation of cardiac automaticity [17-20] and SAN arrhythmias [19, 21]. The postegenomic approach to the study of the heartbeat has a great importance for different reasons. First, the exact ionic mechanism underlying the initiation of automaticity has not been entirely elucidated and different views of the relative importance of ionic channels in the generation of pacemaking have been proposed [10, 22-24]. Understanding the mechanisms of cardiac pacemaking is also important for elucidating the basis of idiopathic inherited diseases of the heart rhythm, and for the pharmacological management of cardiac ischemia and heart failure [25]. Finally, a cellular therapy approach to heart disease employing adult stem cells (ESC) to generate biological pacemaker in situ is starting to emerge [26, 27]. Here, we will discuss the role of ionic channels in the generation of pacemaker activity. We will first review the ionic channels expression pattern in pacemaker cells and the possible mechanisms linking the activity of ionic channels and intracellular Ca2+ release to automaticity and its regulation by the autonomic nervous system. We will then discuss how ionic channels involved in pacemaker activity can serve as useful targets for the development of new therapies for the management of cardiovascular diseases. CELLULAR BASIS OF CARDIAC PACEMAKER ACTIVITY The electrical hallmark of pacemaker cells is the ability to generate spontaneous periodical oscillations of the membrane potential. These cells act as biological clocks initiating the cardiac impulse and driving the contraction of myocardial contractile cells. During the adulthood, heart automaticity is generated by a relatively small population of pacemaker cells located in the sino-atrial node (SAN). The generation of automaticity in pacemaker cells is due to the presence of the diastolic depolarisation (or pacemaker depolarisation), a slow depolarising phase of the action potential, which leads the membrane potential at the end of an action potential to the threshold of the following action potential [28]. The diastolic depolarisation develops during the diastole of the cardiac contraction cycle and constitutes an intrinsic property of cardiac cells endowed of automaticity. In the early embryo, myocardial cells are also capable of generating pacemaker activity, thereby controlling their own contraction rhythm. After the development of the cardiac conduction system, the working myocardium loses its pacemaking properties and the SAN takes over the chronotropic control of the heartbeat [29]. The SAN region is formed by a heterogeneous population of pacemaker cells. Cells located in the periphery of the node show an intrinsic faster rate than cells in the center of the node [30, 31]. However, cells in the center of the node set the overall SAN rate [32], since cells at the periphery of the SAN are electrotonically inhibited by the right atrium [33]. The heterogeneity in the intrinsic firing rate of cells in the center and in the periphery of the SAN is due to a region-

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ally regulated expression of ionic channels [34-38] and Ca2+ handling proteins [39, 40]. SAN cells heterogeneity underlies many integrative properties of SAN pacemaking. The electrophysiological organisation of the SAN region has been recently reviewed in detail [11]. Beside the SAN the atrio-ventricular node (AVN) and the Purkinje fibers network can also generate viable pacemaker activity. Due to their intrinsically higher firing rate, SAN pacemaker cells suppress the pacemaker activity of the rest of the cardiac conduction system and determine the overall heart rate. Even if pacemaker activity of the conduction system in vivo is normally suppressed by the dominant firing frequency of the SAN, it can become dominant in case of SAN failure or block of the atrio-ventricular conduction [41]. For recent review on the functional organisation of the AVN and Purkinje fibers, the reader can consult references [42] and [43]. Spontaneously beating cells are also identifiable in the right atrium [44], in the right and left ring bundle branches, in the mouse Bachmann’s bundle responsible for the conduction of the impulse to the left atrium (Mangoni, unpublished observations) and in the tricuspid valve [45]. Pacemaker activity in the bundle branches and in the Bachmann’s bundle can be linked to the derivation of this tissue from the embryonic cardiac conduction system [46]. The physiological significance of automaticity in latent pacemaker cells of the right atrium is unclear. These cells have been proposed to be capable of assuming the control of the heartbeat in case of SAN failure and to play a role in the generation of atrial ectopic beats triggering atrial fibrillation [44]. In the left atrium, cells generating focal automaticity have been found at the boundary between the left atrium and the pulmonary veins in human subjects affected of paroxystic atrial fibrillation [47]. The key role of these cells in triggering and sustaining atrial fibrillations has been clearly recognized [48, 49]. IONIC CHANNELS IN SPONTANEOUSLY BEATING CELLS As indicated above, the expression pattern of ionic channels in pacemaker cells has been studied by the patch-clamp technique. These studies have provided a comprehensive picture of the ionic currents involved in automaticity [10, 11]. Electrophysiological data have been recently implemented by a large scale, comparative study of the differential expression profile of ionic channels subunits between the mouse SAN, AVN and that of the working myocardium at the mRNA level [50]. Several recent reviews have discussed the physiology of cardiac ionic channels [43, 51]. Here, we will focus on the current knowledge about the ionic channels in pacemaker cells and highlight some differences in the ionic channel expression pattern of cardiac cells endowed of automaticity and myocardial cells devoted to contraction. Hyperpolarization-Activated f-Channels The electrophysiological marker of pacemaker cells is the hyperpolarization activated “pacemaker” current (If). If has

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been found in all mammalian automatic heart cells coming from the SAN and the conduction system investigated thus far [23] and has been proposed to constitute the primary ionic mechanism initiating cardiac pacemaking [23, 25, 52]. If is strongly expressed in the SAN [53, 54], yet its density is maximal in larger cells of the SAN periphery [35]. If is also detectable in the working myocardium, even if at low density [55]. In the ventricle, If displays very negative activation range, well below the physiological myocardial resting potential [56]. It has been shown that heart disease have substantial effect on the myocardial If density. Indeed, hypertension, chronic atrial fibrillation and heart failure are all factors that have been shown to enhance If expression and to positively shift current activation in the myocardium from animal models and humans [57-61]. If is a mixed cationic current carried by Na+ and K+ ions [62]. Under physiological conditions the main trasmembrane flux through f-channels is due to Na+ ions which have higher permeability than K+ [63]. If is activated upon membrane hyperpolarization and supplies inward current in the voltage range corresponding to the diastolic depolarisation [64]. Recently, a measurable Ca2+ permeability of f-channels formed by recombinant HCN2 channels (see below) has been identified [65]. Intracellular Ca2+ ions have been reported to positively regulate If by either increasing total conductance [66] or by shifting the current voltage-dependence of activation to more positive voltages [67]. The exact mechanisms by which Ca2+ ions regulate If has not been elucidated, previous evidence obtained in inside-out patches seem to exclude a direct effect on the channel protein [68]. In intact pacemaker cells, If is regulated in opposite ways by catecholamines and acetylcholine (ACh). Stimulation of the -adrenergic receptors robustly stimulates If [53], while muscarinic receptors activation inhibits If [69]. The autonomic regulation of If is due to direct activation of If channels by intracellular cAMP [70, 71]. Particularly, cAMP accelerates activation of f-channels and facilitates opening at a given voltage [72]. A gene family coding for If channels has been cloned from the mouse [7375], rabbit [76] and human tissue [77-79]. Four isoforms named HCN1-4 have been cloned and expressed in heterologous system [80]. These isoforms differ in their voltage dependence for activation and relative sensitivity to cAMP [80, 81]. Consistently with the view of a dual allosteric regulation of HCN channels by voltage and cAMP [82, 83], HCN1 channels have fast activation kinetics and low sensitivity to cAMP [74, 75], while HCN4 channels have slow activation kinetics and strong sensitivity to cAMP [76-78]. As to HCN2 channels, they display intermediate gating properties between HCN1 and HCN4 channels [78, 84]. Also the kinetics of HCN channels can be regulated under some experimental conditions by the short transmembrane protein KCNE2 [85, 86]. Specifically, KCNE2 has been shown to enhance the surface expression and kinetics of recombinant HCN2 channels in heterologous expression system [85, 87] and in cardiac myocytes [86]. It has also been proposed that KCNE2 can regulate recombinant HCN2 channel opening from timedependent, to time-independent, thus switching the macroscopic HCN2 to an instantaneous component [87, 88]. In the

heart, HCN4 constitute the major isoform coding for If in the SAN [89, 90] and the AVN [50]. Expression of the HCN1 and HCN2 isoforms has been found in the SAN [50, 82, 8991]. The working myocardium expresses moderate levels of HCN2 and HCN4 mRNA [50, 89]. The exact subunit composition of If channels in the SAN has not been elucidated. The robust SAN expression of mRNA coding for HCN4 channels, together with the strong sensitivity of native If channels to cAMP [72], indicates that HCN4 is the major molecular determinant of native If channels. Nevertheless, native If channels display faster activation kinetics than do recombinant HCN1-HCN4 heterotetramers, a difference which cannot be compensated by intracellular cAMP or coexpression of KCNE2 [82]. This observation indicates additional and yet unidentified factors contribute to the setting of the native If kinetics and voltage dependency. Voltage-Dependent Ca2+ Channels (VDCCs) The major pathway for Ca2+ entry in cardiac pacemaker cells is through VDCCs. Pacemaker cells express at least two distinct families of VDCCs, the L- and T- type Ca2+ channels [54, 92-94]. L-type Ca2+ channels are widely expressed throughout the cardiac tissues, are sensitive to antagonist and agonist dihydropyridines (DHPs) such as nifedipine and BayK-8644 and are positively regulated by protein kinase A (PKA) -dependent phosphorylation. Excellent reviews on the tissue distribution, physiology and pharmacology of L-type Ca2+ channels have been recently published [95, 96]. The electrophysiological study of heart tissues has allowed the description of the biophysical properties of VDCCs in cells coming from the SAN, the conduction system and the working myocardium [43, 51]. The fundamental role of L-type VDCCs in the initiation of myocardial contraction is well established [97, 98]. Cardiac L-type channels activate upon membrane depolarisation with variable threshold between 50 and -30 mV and display mixed Ca2+- and voltage-dependent inactivation [19, 92, 99-101]. In the SAN, the density and kinetics of ICa,L is also regulated by both PKA [102] and activated Ca2+/calmodulin –dependent protein kinase II (CaMKII) which regulates activation and reacti-vation kinetics [103]. In ventricular cells, CaMKII facilitates L-type channel opening in response to membrane depolarisation or Ca2+ entry, thereby modulating ICa,L amplitude and inactivation [104-106]. L-type Ca2+ channels are constituted by a central pore forming 1 subunit associated with different accessory subunits (2, and ) [95]. To date, four different L-type 1 subunits have been cloned and form the Cav1 family. L-type Ca2+ channels are highly sensitive to DHPs. The Cav1.1 1 subunit initiates contraction of the skeletal muscle [107]. Cav1.4 is expressed in the retina, spinal cord and immune cells [108, 109]. In contrast, Cav1.2 and Cav1.3 are expressed in the brain, the cardiovascular system and neuroendocrine cells [110]. In the heart, the Cav1.2 is the major molecular determinant of the cardiac ICa,L. The Cav1.3 subunit has been first considered as specific of neurons [111, 112] and neuroendocrine cells [113]. However, it is also expressed in the atrium and the SAN [19, 50, 114, 115]. Compared to Cav1.2,


the Cav1.3 1 subunit forms L-type channels having lower threshold for activation and lower sensitivity to DHPs. Also, Cav1.3 channels show slower inactivation kinetics than Cav1.2 channels [116, 117]. T-type channels activate at more negative voltages than L-type channels and display typical slow criss-crossing activation and fast voltage-dependent inactivation. In addition, T-type channels have smaller single channel conductance than L-type channels [118]. Three distinct 1 cDNAs named Cav3.1, Cav3.2 and Cav3.3 code for T-type channels and form the Cav3 gene family. T-type channels have been cloned from the rat, mouse and humans [119-124], by searching molecular databases for expressed tag sequences (EST) coding for novel Ca2+ channels 1 subunits. The Cav3.2 isoform has been cloned from a human heart library [122] and indicated as an important molecular determinant of the cardiac ICa,T. However, the Cav3.1 isoform is functionally expressed in the atrial AT-1 cell line [125] as well as in the developing mouse heart [126]. Furthermore, measurements of the expression of Cav3 1 subunit in the adult mouse SAN by in situ hybridisation, showed that the expression of Cav3.1 is much stronger than that of Cav3.2 [115]. It has been recently shown that the Cav3.2 isoform constitutes the predominant molecular determinant of ICa,T in the mouse embryonic heart. However, the expression of the Cav3.1 subunit increases during the perinatal period and becomes dominant after birth and the adulthood [127]. No expression of the Cav3.3 isoform has been found in the heart [124]. We presently lack appropriate pharmacological tools to separate the contribution of Cav3.1 and Cav3.2 in the generation of cardiac ICa,T. Nevertheless, Cav3.2 channels are more sensitive to block by Ni2+ ions than Cav3.1 channels, the concentration for halfblock (EC50) being about 5 M and 150 M for Cav3.2 and Cav3.1 channels respectively [128]. This property helps discriminate Cav3.1 from Cav3.2 –mediated ICa,T in some conditions. Voltage-Dependent Na+Channels Voltage-dependent Na+ channels are expressed in SAN pacemaker cells in a variable and possibly species-dependent way. In cell culture conditions, a significant fraction of SAN pacemaker cells express the fast sodium current (INa) [129, 130]. As for other ionic channels, INa is heterogeneously expressed in the adult rabbit SAN [11, 31]. INa expression is higher in large cells (presumably from the periphery of the SAN) than in small cells (presumably from the SAN center) [31]. Consistently with the heterogeneous expression of INa in isolated SAN cells, pacemaking in the periphery of the SAN is slowed by block of INa by tetrodotoxin (TTX), as opposed to pacemaking in the center of the SAN which is insensitive to TTX application [34, 131]. Compared to the adult SAN, pacemaker cells of neonatal rabbits show constitutive expression of INa [132]. The expression of INa is maximal during the first three weeks after birth, and then declines irrespectively from the cell size in young individuals to reach its minimum in the adult [132]. Constitutive expression of INa has also been observed in adult mouse cells

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[54, 133, 134]. This up-regulation of INa in SAN pacemaker cells can explain in part the high heart rate in the mouse. The SAN INa is pharmacologically distinct from INa expressed in working myocytes, since it shows higher sensitivity to TTX. Indeed, a TTX-sensitive INa is recorded, in rabbit neonatal [135] and in adult mouse pacemaker cells [134]. Consistently with this view, nanomolar concentrations of TTX are sufficient to induce a significant slowing of pacemaking in isolated mouse hearts [136] and SAN pacemaker cells [134]. It has thus been proposed that SAN pacemaker cells express distinct “TTX-sensitive“ isoform(s) of Na+ channels genes with respect to the working myocardium. Indeed, the expression of the neuronal Nav1.1 isoform has been shown in neonatal rabbit SAN cells at the mRNA level by in situ hybridisation [135]. Expression of Nav1.1 has also been found in the mouse SAN by immunohistochemistry [134]. Sustained Inward Channels (Ist) The presence of a Na+ and voltage-dependent inward current activated upon depolarisation has been reported in pacemaker cells and has been named Ist [137]. Ist is activated at negative potentials of about -70 mV, peaks at about -50 mV and is enhanced by the -adrenergic receptor activation [137]. Ist is clearly distinct from INa, since it is insensitive to TTX, is blocked by DHPs antagonists and is partially inhibited by divalent cations, including Ca2+, Mg2+ and Ni2+ [137]. Ist has been found in SAN pacemaker cells from the rabbit, guinea pig, rat and the mouse [137-140]. Single st-channels are permeable to Na+, have a unitary conductance of 13 pS and, similarly to L-type channels, are facilitated by the DHP agonist Bay-K 8644 [141]. The presence of Ist seems to be specific of spontaneously active SAN and atrio-ventricular node cells, since no Ist has been recorded in quiescent cells from these two regions [142]. The molecular nature of Ist has not yet been determined. It has been proposed that the Ist current is generated by a new subtype of L-type channels [137]. This hypothesis would be consistent with the pharmacological sensitivity of Ist to agonist and antagonists DHPs . Voltage-Dependent K+ Channels in Pacemaker Cells Three major voltage-dependent K+ currents have been identified and studied in SAN pacemaker cells, the fast (IKr) and slow (IKs) components of the delayed-rectifier and the transient outward current (Ito). For a detailed review on the structure, function and physiopathology of these channels in the heart, the reader is referred to some excellent recent reviews [51, 143-146]. In spontaneously active cells, these currents control the action potential repolarization rate. In isolated SAN pacemaker cells, IKr is activated upon depolarisation from about -50 mV, peaks between -20 and 10 mV and display inward rectification [147-150]. Membrane depolarisation speeds both activation and inactivation of IKr . For membrane potential positive to 10 mV, current inactivation progressively counterbalances activation, generating a region of negative slope conductance in the IKr current-tovoltage relation [150]. When the membrane potential is



switched back to negative potentials, IKr deactivation tail currents are recorded. Tail currents are due to channel fast recovery from inactivation and consequent re-opening, followed by time-dependent closure at negative membrane potential [147]. IKr is specifically blocked by class III methanesulfonanilide compounds E-4031 and dofetilide (UK-68798), which are commonly employed to study the functional role of this current in the heart. Furthermore, IKr is sensitive to many different compounds used in the current medical practice [144]. IKr channels are coded by the ERG gene family, which constitute the mammalian homologue of the ether-ago-go gene family of Drosophila melanogaster. Three members of the ERG gene family have been cloned and named ERG1-3. The ERG1 gene is expressed in the heart. Mutations in the human ERG gene or in its accessory subunit KCNE2 can lead to the long-QT syndrome and/or abnormalities of ventricular repolarization [151]. In the mouse SAN, three isoforms of the ERG1 gene namely 1a, 1a’ and 1b have been found at the mRNA level [150]. The contribution of different isoforms of ERG1 in the formation of the native cardiac IKr is matter of debate. It has been proposed that both ERG1a and 1b are able to form recombinant channels with properties similar to those of native IKr [152]. This view has been challenged by recent evidence indicating that only the ERG1a channel protein is detectable in western blots from mammalian working myocardium [153]. The contribution of ERG1 isoforms to the generation of native SAN IKr channels remains to be elucidated. The IKs current is distinguished from IKr by its slower kinetics of activation and faster deactivation as well a by specific block by the 293B channel blocker [38, 154, 155]. Three genes coding for IKs KCNQ1-3 have been cloned, but only the KCNQ1 gene seems to be expressed in the heart [143]. The expression of both IKr and IKs show heterogeneity in the rabbit SAN. large SAN cells show higher IKr and IKs density [38]. Species-dependent differences also exist as to the expression of IKr and IKs. For example, IKr has not been recorded in pig SAN cells which seem to express only IKs [156]. Also IKs has not yet been recorded in mouse SAN cells (Mangoni, unpublished observations). Taken together, these reports indicate that the exact ionic and molecular composition of IK in the SAN can vary according to the cellular organisation of the pacemaker tissue and/or the species considered. The contribution of IKr and IKs to -adrenergic regulation of pacemaker activity would need further investigation. Indeed, early studies employing multicellular SAN preparations have shown that adrenaline strongly enhances IK [6, 157]. These observations have been confirmed in isolated rabbit pacemaker cells [158]. Sensitivity of IKr to -adrenergic agonists has been shown in ventricular myocytes [159], but this issue has not been yet investigated in SAN cells. The hallmark of Ito current is its fast activation and inactivation kinetics upon cell depolarisation (see for example [160]). Ito is also pharmacologically distinguished from other K+ currents for its sensitivity to 4-amino pyridine (4-AP). Two major, kinetically distinct components of Ito have been identified and named Ito,f and Ito,s, according to their fast and

slow inactivation kinetics. As for IKr and IKs, the expression of Ito in SAN pacemaker cells is heterogeneous [36, 37]. Particularly, automaticity of SAN tissue from the periphery of the SAN is more sensitive to block of Ito by 4-AP than that from the center of the SAN [36]. This observation is reflected in single cells by a higher expression of Ito in large cells presumably from the SAN periphery as compared to small cells which are predominant in the center of the SAN [37]. Ito channels are coded by Kv1 and Kv4 gene families [161]. Gene inactivation of the Kv1.4 subunit selectively abolishes Ito,s [162]. The cardiac Ito,f is formed by heteromultimers of the Kv4.2 and Kv4.3 subunit [163]. Ito channel complex is also associated to KChIP proteins [164]. These subunits constitute an integral part of the channel complex and regulate the channel inactivation kinetics and expression to the cellular membrane [164]. In the heart the KChIP2 protein is associated to Kv4.2 and Kv4.3 channel complexes [163]. Inactivation of the KChiP2 protein by gene inactivation in the mouse yields to abolition of Ito in myocardial cells, prolongs the action potential duration and induces ventricular tachiarrhythmias [165]. Voltage-Independent K+ Channels in Pacemaker Cells The acetylcholine-activated current (IKACh) is robustly expressed in both the SAN and the atria [166-168]. IKACh is stimulated by activation of the muscarinic or adenosine receptors which are coupled to G-proteins [169, 170]. IKACh channels are then directly open by binding of the activated subunits to the channel complex [171, 172] (see Fig. 2). A gene family coding for IKACh channels, Kir3 has been cloned. Among the four members of this family, Kir3.1 and Kir3.4 are expressed in the heart [173]. IKACh channels are formed by tetramers containing both Kir3.1 and Kir3.4 channels [174]. Importantly, the Kir3.1 channel protein requires the presence of another functional member of the Kir3 family to be targeted to the cell membrane [175]. As a consequence, mice lacking Kir3.4 channels show no cardiac IKACh [176]. It has recently been shown that rabbit SAN cells express the ATP-dependent K+ current (IK,ATP) [177]. This current is activated when the intracellular concentration of ATP is lowered and slows down pacemaking by hyperpolarising the maximum diastolic potential. The physiological significance of IK,ATP in the SAN has not yet been elucidated. One possibility is that this current slows the heart rate under ischemic conditions, thus contributing to protect the myocardium from stunning [177]. It is a classical concept that spontaneously beating SAN and AVN cells have low or no detectable expression of the inward-rectifier K+ current IK1 [6, 10, 178, 179]. In contrast, this current is robustly expressed in non-pacing working myocardium [51] and in the Purkinje fibers network (see for example [7]), thus accounting for the negative diastolic potential recorded in these cells as compared to spontaneously active myocytes, which show a more positive maximum diastolic potential. Nevertheless, rat and mouse SAN express IK1, even if at a much lower density than the working myo-


cardium [139, 140]. Cardiac IK1 channels are coded by the Kir2.1 and Kir2.2 channel subunits [143]. DIFFERENTIAL EXPRESSION OF IONIC CHANNEL GENES IN PACEMAKER TISSUE AND THE WORKING MYOCARDIUM The mechanism by which the genetic program controls the differential ionic expression pattern in spontaneously active cells and in working myocytes has not been elucidated. A better knowledge of the expression pattern of channel subunit in pacemaker tissue is of great importance for understanding the ionic basis of automaticity and arrhythmias. In a recent collaborative study involving three different groups, we have developed a high-throughput approach capable to simultaneously assess the expression pattern of the ionic channel repertoire of mouse pacemaker tissue and the working myocardium [50]. Such an approach is possible by taking advantage of high density real-time RT-PCR array technology, which allows handling of small tissue samples such as the SAN and AVN which contain low quantities of mRNA. By using large-scale real-time RT-PCR, we have profiled 71 channels and related genes in the SAN, AVN, the right atrium (RA) and the left ventricle (LV). The expression profile of transcripts discriminating each region of the heart is depicted in Fig. (1). Genes that are predominantly expressed in the nodes or in the myocardial chambers are listed below each column (see figure legend). subunit genes together with their corresponding subunit(s) (arrows) are classified in Fig. (1). From this comparison, it appears that HCN1 and HCN4 transcripts display the highest expression level in the SAN. High expression of Cav1.3 and Cav3.1 tran-

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scripts is also found in the SAN and the AV node. Also HCN2 display expression in the SAN, even if at lower levels compared to HCN1 and HCN4. These results highlight the transcriptional link of these channels to the pacemaker function [19, 180, 181]. Concerning the AVN, the voltage-gated Na+ and K+ channel subunits Nav1.7, Kv1.6, the subunit Kv1 are specific for this node. Significant expression of Nav1.1 has also been found in the AVN. Among the Ca2+ channels families, Cav1.3 and Cav3.1 are more specific for the nodes and Cav22 constitute a potential accessory subunit for these Ca2+ channels in the nodes. A possible association between Cav3.1 and Cav22 subunit has been indicated by Gao and co-workers [182]. This study has shown an increase of 176% of the Cav3.1 current amplitude upon coexpression of the Cav22 subunit. Among the voltagegated Na+ channel - and -subunits, pacemaker tissues distinguish themselves by the predominant expression of Nav1 and Nav3 (Fig. (1)). If we consider that Na+ channel -subunits exert multiple effects including modulation of channel gating, facilitation of channel trafficking and regulation of cell adhesion [183], the association of Nav1 and Nav3 can speed the inactivation of Na+ channels subunits, to adapt their function to the fast basal heart rate of the mouse. The expression profile of K+ channels also show regional specificity. Kv1.1, Kv1.4, Kv1.6, Kv1 and Kv3 are up regulated in the nodes. We can speculate that Kv1.1 and Kv1.6 could play a role in the action potential repolarization in pacemaker cells, in association with the Kv1 and Kv3 subunits respectively. Electrophysiological investigation of mouse SAN and AVN cells is needed to elucidate the functional role of these K + channels in pacemaking and conduction.

Fig. (1). Properties of the ionic channels and transporters expression pattern in the SAN, AVN and the working myocardium. Each column identifies ionic channels subunits that are highly or specifically expressed in the tissue considered. Genes showing less expression in a given tissue compared to dominant genes are indicated in smaller letters. Relatively low expression of KCNE2 mRNA was found in the mouse SAN (not indicated). Arrows indicate the possible interactions between different ionic channels and subunits.



Fig. (2). The cellular organisation of some ionic channels in an ideal SAN pacemaker cell is shown here. Ionic channels are indicated with different colours and named according to the ionic currents they are known to underlie. Also, the name of the gene coding for given ionic channels is indicated when identified. Filled black diamonds represent Na+ ions, blue diamonds indicate K+ ions and red circles stand for Ca2+ ions. The key proteins composing signalling pathways involved in autonomic regulation of pacemaker activity are depicted. Noradrenaline (Ndr), ACh and adenosine (Ado) receptors are shown together with stimulatory (s) and inhibitory G-proteins (i). The NO-dependent muscarinic pathway for regulation of ICa,L has been neglected for clarity. cAMP (violet pentagons) is produced by AC activity and binds to the regulatory subunits (R) of PKA. Interaction between PKA and ionic channels has been indicated according to the current literature (see text). Two specialized signalling compartments are suggested. First, regions of the cellular membrane containing high density of f-/HCN channels are indicated. Regulation of cAMP production is also suggested in the vicinity of HCN channels. Subsarcolemmal Ca2+ signalling space integrating VDCCs and NCX with the SR-RyRs regulating SR Ca2+ release is also shown.

MECHANISMS OF PACEMAKING The Concept Pacemaking: The Generation and Regulation of the Slow Diastolic Depolarisation The exact nature of the mechanism underlying the generation of automaticity in primary pacemaker SAN cells has not been completely clarified. Beside ionic channels, pacemaking also depends from the activity of the Na+/K+ pump and the Na+/Ca2+ exchanger (NCX). Research groups in the field have studied and emphasized the role different ionic currents (such as If, ICa,L, IKr and Ist) or NCX and intracellular diastolic Ca2+ release in the initiation of the pacemaker potential [8, 22, 23, 64, 92, 184-187]. Here, we wish to stress that an attentive look to experimental data in the current literature can lead to the conclusion that all these apparently different proposals are not mutually exclusive and that the proper generation and regulation of the diastolic phase in different physiological situations requires the intervention of more than one ionic mechanism. The generation of automaticity necessitates the activation of an inward current at the end of the repolarization phase for initiating the diastolic depolarisation [23]. For our discussion, we will adopt the working hypothesis that upon decay of IKr, the diastolic phase is generated by the association of If, together with Ist, ICa,T ICa,L and INa. We will now review the current evidence supporting the contribution of these volt-

age-dependent channels to automaticity. Also, we will discuss the involvement of diastolic release of Ca2+ from the sarcoplasmic reticulum (SR) in the regulation of SAN pacemaking. Most of these data come from the rabbit SAN and have been obtained by pharmacological manipulation of the cellular action potential cycle and ionic channels. To investigate the mechanism regulating the cardiac pacemaking it is also important to provide genetic evidence associating the activity of a given ionic channel to specific dysfunction of cardiac automaticity both in vivo and in vitro. The possibility to investigate SAN pacemaking in genetically-modified mouse strains is now providing exciting and sometimes unexpected new insights in the physiology of the heart rhythm and rate. The mouse SAN has constituted a remarkable technical challenge. Indeed, due to the tiny size of the dominant pacemaker region in the mouse heart [188], the possibility to obtain viable spontaneously-beating cells for electrophysiological recording has been considered as low. Our group [54, 133] has been the first to isolate mouse pacemaker cells by adapting the method used to isolate rabbit SAN cells [189]. Soon after our report, other groups have successfully employed this new experimental model to study the mouse cardiac automaticity and ionic currents in both wild-type [134, 140, 150, 190, 191] and genetically modified mouse strains [19, 101, 192]. More generally, the new possibility of isolating mouse SAN tissue and primary pacemaker cells has cre-


ated a new interest into the study of the ionic mechanisms of the generation and regulation of cardiac pacemaker activity. ERG Channels and Pacemaking As indicated above, ERG channels underlie IKr. This current controls the rate of action potential repolarization in SAN cells and is one of the major determinants of the cell maximum diastolic potential [149, 150, 193]. As a consequence, IKr influences the way in which voltage-dependent ionic channels activate in the diastolic depolarisation [150]. Accordingly, partial block of IKr in isolated SAN cells robustly reduces pacing rate, or can eventually stop automaticity [149, 150, 193]. Block of IKr by E-4031 also stops pacemaker activity in isolated SAN tissue strips [194]. However, block of IKr does not suppress automaticity in isolated intact hearts [150] or in atrial-SAN tissue preparations [194]. These observations have been accounted for the electrotonic load imposed to pacemaking tissue by the adjacent atrial tissue, which can partially drive SAN repolarization upon IKr inhibition [194]. IKr thus play a fundamental role in the way in which SAN automaticity is coupled to the surrounding atrial tissue. Furthermore, IKr is differentially expressed in the SAN [195], small cells display less IKr density than larger cells [196]. Differential expression of IKr can explain the higher sensitivity of automaticity in the center of the SAN to E-4031. f-Channels and Pacemaking Three main lines of evidence indicate that the If current constitutes a key element in the initiation and control of the diastolic depolarisation. First, If is activated in the diastolic depolarisation range close to the maximum diastolic potential [8, 64, 197]. Secondly, all known pharmacological inhibitors of f-channels induce negative chronotropism in SAN tissue and isolated pacemaker cells [35, 198]. To this respect, the heterogeneous regional distribution of If influences the relative sensitivity to different f-channels blockers of pacemaker activity in the center versus the periphery of the SAN. Indeed, the negative chronotropic effect of Cs+ and UL-FS 49 is maximal at the periphery of the SAN and less pronounced in the center [35]. Third, insights from the zebrafish [17] and genetically modified mouse strains [181, 192] have shown that inactivation of f-channels induces dysfunctions of pacemaker activity. The first genetic evidence associating f-channels to the generation of cardiac pacemaking came from a large scale mutagenesis study in zebrafish. In this study, mutant strains were selected for mutations affecting the cardiovascular system. Upon selection of embryos, the slow mo (smo) mutant line displayed reduced heart rate [17]. Isolated heart cells from smo embryos display strong down regulation of If and abolition of the fast kinetic component of this current [17]. The association between the loss of If fast component and bradycardia in smo mutants constitute strong evidence of the importance of f-channels in zebrafish pacemaker. Inactivation of HCN2 channels in the mouse is associated with sino-atrial arrhythmia and down regulation of If in SAN

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cells by about 30% [192]. HCN2 inactivation slows the kinetics of If activation, indicating that HCN2 channels can be associated with the generation of a fast component of If in the SAN. However, the maximal If current stimulation by cAMP is not changed in SAN cells from HCN2 knockout mice [192], suggesting that the major channel responsible for the If-mediated stimulation of heart rate is HCN4. HCN2 knockout mice is not bradycardic, indicating that these channels are associated with the stabilisation of the SAN rate. As opposed to HCN2, inactivation of HCN4 channels provokes lethality in mouse embryos. Particularly, mice lacking HCN4 channels globally, or in a heart specific way, die between day 9 and 12 postcoitum [181]. Embryo lethality is associated with slow heart rate, almost complete abolition of If and insensitivity of the heart rate to cAMP [181]. One of the most interesting aspects of the phenotype of HCN4 knockout mouse embryos is the lack of mature pacemaker cells in the developing heart. Indeed, only cells characterised by an early embryonic pacemaking [199] are found in HCN4 deficient hearts. This observation is strongly suggestive of a contribution of HCN4 channels in the development of the heart conduction system [181]. VDCCs in the Generation of Diastolic Depolarisation in SAN Pacemaker Cells ICa,L in the SAN In the SAN, ICa,L density is positively correlated with the cell size, a phenomenon which is reflected at the SAN tissue level with a differential contribution of ICa,L to the pacemaker action potential. ICa,L plays an obligatory role in pacemaking in the center of the rabbit SAN, since abolition of ICa,L by nifedipine blocked automaticity in this region [34]. In contrast, spontaneously beating tissue from the periphery of the SAN showed only moderate negative chronotropism in the presence of nifedipine, [34]. These results are consistent with the view that the pacemaker action potential upstroke in the center of the rabbit SAN depends from ICa,L,, while in SAN periphery INa is robustly expressed and compensates for ICa,L blockade. The differential role of ICa,L in the center and the periphery of the SAN underlines the problem of separating the possible contribution of ICa,L to the diastolic depolarisation from that of the upstroke phase of the action potential. Indeed, blockade of ICa,L during the action potential upstroke will also affect the recruitment of ionic currents during repolarization as well as the following diastolic depolarisation. In the SAN, the activity of L-type VDCCs is regulated by basal phosphorylation state of the channel which depends from both PKA [102] and activated Ca2+/calmodulin –dependent protein kinase II (CaMKII) [103]. It has been shown in ventricular cells that CaMKII facilitates channel opening in response to membrane depolarisation or Ca2+ entry, thereby modulating ICa,L amplitude and inactivation [104-106]. In the SAN, an intact activity of CaMKII is required for automaticity, since CaMKII inhibitors stop pacemaking [103]. The essential role for CaMKII in automaticity has been explained by the regulation of ICa,L activation and reactivation kinetics by CaMKII [103]. Verheijck and co-workers [100] have recorded the net DHP-sensitive ICa,L at different times during



the diastolic depolarisation and shown that ICa,L is activated first during the early diastolic depolarisation, and then progressively increases up to the threshold of the action potential upstroke. The observation that inhibition of ICa,L by DHPs induced bradycardia in vivo in anaesthetised mice [200] constituted a first evidence of the implication of L-type channels in the automaticity of the mouse heart. However, the major breakthrough in the study of the role of VDCCs in cardiac pacemaking was the observation that mice in which the gene coding for Cav1.3 Ca2+ channels has been inactivated showed major sinus node dysfunctions [18]. Two independent studies have subsequently demonstrated that Cav1.3 channels play an important role in pacemaking both in vivo [101] and in vitro at the cellular level [19, 101]. Cav1.3 channels have been shown to significantly contribute to the diastolic depolarization. More generally, one of the most important conclusions coming from these studies has been that Cav1.3 channels are specifically linked to the regulation of pacemaking and generate ICa,L with distinct properties with respect to that of Cav1.2 ICa,L channels that are devoted to the initiation of myocardial contraction. Cav1.3 knockouts mice are characterized by bradycardia associated with strong sinoatrial arrhythmia. Arrhythmia and bradycardia are still observed in vivo after pharmacological block of the autonomic input [18]. The evidence that, isolated atria [18] and SA nodes [101] of Cav1.3 knockout mice still beat slower than their wild-type counterparts, further reinforces the idea that a dysfunction in the automaticity of the pacemaker cell was responsible for the observed in vivo cardiac phenotype. This has been confirmed by patch-clamping of SAN pacemaker cells from Cav1.3 knockout mice, which show erratic pacing rate and slower automaticity than wild-type cells [19]. Cav1.3 knockout mice also have defects of the atrio-ventricular conduction [18, 101, 201]. Particularly, Cav1.3 knockout mice show I and II degree atrioventricular blocks, an observation which has been found in both freely moving [18] and anesthetized mice [101], as well as in isolated Cav1.3 knockout hearts [201]. The characterisation of ICa,L in wild-type and Cav1.3 knockout pacemaker cells has been rich in surprising results, and led to the identification of Cav1.3 channels as an important ionic mechanism underlying the diastolic depolarisation. Particularly, we have shown that the lack of Cav1.3 channel in SAN cells from knockout mice abolishes all ICa,L in the voltage range corresponding to the diastolic depolarisation [19]. Consistently with the conclusion that Cav1.3 – mediated ICa,L is selectively abolished by gene inactivation, ICa,L in pacemaker cells from knockout mice showed augmented sensitivity of ICa,L to DHPs [19] as well as accelerated inactivation kinetics [101]. The threshold for activation of Cav1.3 channels in mouse SAN cells in basal conditions is around -50 mV. Under cell superfusion by noradrenaline, the threshold is shifted to about -55 mV [19]. These values are consistent with the observed threshold of the nifedipinesensitive ICa,L measured in spontaneously beating rabbit SAN cells [100], suggesting the existence of Cav1.3 channels also in rabbit SAN cells. Even if the responsiveness of cellular automaticity to cAMP has not yet been investigated in detail in Cav1.3 knockout pacemaker cells, it is interesting to note that the maximal rate of Cav1.3 knockout hearts measured in

the presence of isoproterenol is slightly but significantly slower than that of wild-type hearts [201]. This is not surprising if we consider that no activation of ICa,L in the voltage range corresponding to that of the diastolic depolarisation is observed in Cav1.3 knockout pacemaker cells, even under stimulation of the residual Cav1.2 –mediated ICa,L by saturating concentrations of noradrenaline [19]. The cardiac phenotype of Cav1.3 knockout mice, and the observed properties of ICa,L expressed in SAN cells after disruption of the Cav1.3 gene are leading to a revision of the concept of the “cardiac” ICa,L, and now, a distinction between a “pacemaker” ICa,L and ICa,L controlling myocardial contraction seems to emerge. Indeed, since the expression of Cav1.3 channels is restricted to the SAN, the AVN and the atrium, the abolition of Cav1.3mediated ICa,L has a major impact on pacemaking and conduction, but has no effect on the cardiac contractility which is governed by Cav1.2 channels [19, 201]. The view of a functional separation between Cav1.3 and Cav1.2 channels in controlling the heartbeat has been reinforced by employing a mouse strain in which Cav1.2 channels have been rendered insensitive to DHPs by knocking-in a point mutation in the Cav1.2 DHP binding site (Cav1.2DHP-/- mouse). The in vivo bradycardic effect induced by DHPs is not changed in Cav1.2DHP-/- mice indicating that the dominant L-type VDCC isoform controlling the diastolic depolarisation in the adult SAN is in fact Cav1.3 [202]. However, knowledge about the timing of the establishment of the mature heartbeat during development with respect to Cav1.2 and Cav1.3 channels would be of remarkable interest. Transcriptional cross-talk between Cav1.2 and Cav1.3 1 subunits does exist. Knockout of the Cav1.2 1 subunit causes lethality of the late mouse embryo. Heart development in early Cav1.2 knockout embryos is paralleled by overexpression of the Cav1.3 1 subunit [203], as well as by up regulation of an L-type Ca2+ current which seems clearly distinct from Cav1.3 ICa,L, since it is still present in Cav1.3 knockout embryonic hearts [204]. It is believed that the overexpression of these channels constitutes a compensatory mechanism for the loss of Cav1.2 channels, even if it is insufficient at the later stage of the development to ensure viability of the embryo. There also exists evidence that could be interpreted in terms of an involvement of Cav1.2 channels in the generation of automaticity in the embryonic heart. Embryonic hearts from a spontaneous zebrafish mutant strain lacking the Cav1.2 1 subunit (Isl mutant) show depressed and erratic atrial beating [205]. Consistently with the role of Cav1.2 in controlling the cardiac contraction, hearts from Isl mutants have no ventricular contraction. The physiological role of Cav1.2 channels in pacemaking of the adult also remains to be established. One possibility is that these channels control the Ca2+ -dependent upstroke phase of action potential. ICa,T in the SAN ICa,T has been consistently found in all cardiac tissues showing automaticity, including the SAN [92, 93], the AVN [206] and Purkinje fibers [207, 208]. Together with If, ICa,T has also been found in the amphibian sinus venosus [209], suggesting a link between ICa,T and cardiac pacemaking


throughout evolution of vertebrates. Importantly, ICa,T is also expressed in the spontaneously beating embryonic myocardium of the mouse [126] and that of the developing zebrafish heart [17]. ICa,T has also been recorded in early embryonic stem cells showing intermediate and mature pacemaker phenotype [199, 210]. In the adult SAN, non-specific blockers of ICa,T affect pacemaking. Indeed, extracellular application of Ni2+ ions [92, 211], tetrametrine [92] and amiloride [10] induce negative chronotropism in isolated SAN pacemaker cells. However, despite pharmacological data indicating a possible involvement of ICa,T in the generation of automaticity, we would need a detailed explanation of how ICa,T is coupled to the diastolic depolarisation. A possible mechanism by which ICa,T can contribute to the diastolic depolarisation has been investigated in cat atrial latent pacemaker cells [212, 213]. In these cells, application of 40 M Ni2+ induces slowing of the late phase of the diastolic depolarisation. This effect is paralleled by a reduction of Ca2+ release from the sarcoplasmic reticulum (SR). Interestingly, Ni2+ does not affect Ca2+ release during the action potential upstroke (systolic Ca2+), but only Ca2+ released during the diastolic depolarisation (diastolic Ca2+). These data indicate that, in latent pacemaker cells, ICa,T activation during the diastolic depolarisation may trigger local Ca2+ signalling generating net inward current due to the consequent stimulation of INa/Ca during the late phase of the diastolic depolarisation [214]. According to this view, there exists a close spatial link between the membrane T-type channels, the ryanodine receptors (RyRs) of the SR and NCX [215] (see Fig. 2). Functional coupling between T-type channels and the SR could also explain evidence indicating that uncoupling of SR Ca2+ release by application of ryanodine induced down regulation of ICa,T [216]. However, no direct evidence for the participation of ICa,T in triggering subsarcolemmal SR Ca2+ release in primary SAN pacemaker cells has been found thus far. Indeed, superfusion of 50 M Ni2+ failed to inhibit the diastolic subsarcolemmal Ca2+ release during sustained pacemaking [24], as well as spontaneous Ca2+ release in arrested pacemaker cells [217]. The discrepancy between the effects of Ni2+ in diastolic Ca2+ release could be due to the existence of a different mechanism for triggering Ca2+ release in SAN pacemaker cells, or to the more negative diastolic potential improving opening of T-type channels in latent pacemaker cells. Despite the relatively low steady-state availability of T-channels at the maximum diastolic potential of SAN cells, ICa,T can possibly be involved in pacemaking in physiological situation where the maximum diastolic potential undergoes spontaneous hyperpolarization (e.g. tonic muscarinic receptor activation) and/or in pacemaking in the cardiac conduction system. Accordingly with this view, loss of Cav3.1 channels by gene inactivation induces bradycardia at rest in both freely-moving and anesthetized mice. ICa,T is completely abolished in SAN pacemaker cells from Cav3.1 knockout and abolition of ICa,T is accompanied by a slowing of the cellular beating rate. Furthermore, these mice also show slowing of the atrioventricular conduction [180]. As opposed to Cav3.1, mice lacking Cav3.2 channels display no alteration in the heart rate, atrioventricular conduction or arrhythmias [218], thus suggesting that Cav3.1 channels specifically contribute

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to the generation of native ICa,T in the adult SAN tissue. Consistently, recent studies have shown that only Cav3.1 related channels are recorded after birth [127, 219]. It will thus be interesting to investigate the differential role of Cav3.1 and Cav3.2 to pacemaking and cardiac conduction during the cardiac development and during the adulthood. INa and Ist in Cardiac Pacemaking Evidence for the contribution of INa to pacemaking have been obtained from the neonatal rabbit SAN and the adult mouse SAN [132]. In isolated rabbit SAN cells, INa is strongly expressed during the first week after birth and then progressively declines to low expression levels after about 30 days [132]. Application of TTX to spontaneously beating pacemaker cells of newborn rabbits strongly slows down pacemaking indicating that Na+ channels are important contributors for determining the diastolic depolarisation rate in these cells [132]. In contrast, SAN cells from adult rabbits show very weak sensitivity to application of TTX accordingly with the low expression of INa at this developmental stage. The unexpected characteristic of INa in newborn rabbit SAN is its high sensitivity to TTX as compared to the classical ventricular INa. This high sensitivity to TTX is due to the expression of the neuronal Na+ channel subunit Nav1.1 in neonatal cells [135]. Expression of this isoform would be then repressed at the adult stage. The mechanism of contribution of the neonatal INa to pacemaking in neonatal rabbit SAN cells has also been investigated [220]. Here, TTXsensitive current flowing during the diastolic depolarisation has been recorded. This current component is generated by the slow inactivation rate of Na+ channels at diastolic potential after the upstroke phase [220]. The physiological significance of the expression of the neuronal Nav1.1-mediated INa in newborn SAN is possibly linked to the faster heart rate in newborn animals than in adults. In mouse SAN pacemaker cells, the expression of INa is constitutive and persists during the adulthood [54, 133, 140]. Experiments in isolated adult mouse hearts have shown that the heart rate is reduced by low doses of TTX [136]. The expression of the neuronal TTX-sensitive subunit Nav1.1 and Nav1.3 channels has been reported in these hearts [136]. These experiments have demonstrated the specific contribution of TTX-sensitive, neuronal INa in mouse cardiac pacemaker activity. In another study, the sensitivity of pacemaking of mouse SAN cells to TTX has been studied [134]. Here, TTX-sensitive and resistant INa components have been identified. The TTXsensitive component has been associated to expression of the Nav1.1 subunit. Block of TTX-sensitive INa by low doses (50-100 nM) of TTX reduces pacing of isolated mouse hearts [221] and slows automaticity of isolated SAN pacemaker cells [134]. TTX-sensitive INa has been shown to be activated during the late phase of the diastolic depolarisation, thereby influencing both the slope of the diastolic depolarisation and the action potential upstroke [222]. TTX-resistant INa has been linked to expression of the cardiac Nav1.5 subunit [134]. TTX-resistant INa contributes to impulse propagation within the SAN, as well as to conduction between the SAN and the atrium [134, 222].



The lack of specific pharmacological tools targeting Ist, together with the still unidentified molecular basis of this current in SAN cells have prevented to directly investigate the role of Ist in cardiac pacemaking. Nevertheless, a significant role for Ist in the control of the diastolic depolarisation rate has been proposed on the basis of numerical simulations of SAN pacemaker activity in the rat [139] or the rabbit [223]. Results from numerical simulations of pacemaker activity indicate the possibility that Ist can sustain pacemaking by virtue of its low threshold of activation and slow inactivation rate. These properties, would allow Ist to be present throughout the diastolic depolarisation [139]. The relative importance of Ist with respect to that of other currents has also been investigated by a numerical modelling approach [223]. It has been concluded that the capability of Ist to affect pacemaking depends from its relative size compared to other currents contributing to pacemaking such as If or INa. As a consequence, in rabbit central SAN pacemaker cells Ist expression will lead to acceleration of the pacing rate in control conditions and under -adrenergic receptor activation. In contrast, pacemaking at the periphery of the SAN would be less affected by the expression of Ist, due to the larger expression of INa and If [223]. In conclusion, numerical modelling predicts a significant contribution of Ist to pacemaker activity. However, elucidation of the molecular nature of Ist is needed to gain new insights into the importance of this current in pacemaker activity. AUTONOMIC REGULATION OF PACEMAKER ACTIVITY The autonomic regulation of the heartbeat is based on the opposite regulation exerted by the sympathetic and parasympathetic nervous system on cardiac automaticity, atrioventricular conduction and myocardial contractility. Cardiac pacemaker activity is subject to a constant and adaptable equilibrium between the sympathetic and parasympathetic nerve endings, equilibrium which depends from the everyday requirements of the organism. Here we will review the current knowledge about the ionic mechanisms underlying the autonomic regulation of SAN pacemaker activity. Ionic Channels and Sympathetic Regulation of Pacemaking The positive chronotropic effect induced by the sympathetic nervous system is initiated by the activation of the adrenergic receptor, which stimulates the enzyme adenylylcyclase (AC) converting ATP in cyclic-adenosine-monophosphate (cAMP), thereby starting the signalling cascade which involves the activation of PKA and other cAMP-dependent processes (see Fig. (2)). As discussed above, cAMP facilitates the opening of f-channels [72], stimulates ICa,L [19] and Ist currents through PKA-dependent phosphorylation [142] (Fig. (2)). cAMP also promotes release of Ca2+ from RyRs of the SR during the diastolic depolarisation (Fig. (2)). PKAdependent phosphorylation of L-type channels induces an increase of the channel mean open time and facilitation of channel opening for a given voltage [224]. If and ICa,L have been proposed to constitute important mechanisms in the

sympathetic control of pacemaking [8, 9, 23, 225]. The capability of If to respond to an even small increase of intracellular cAMP concentration by positively shifting its voltage dependence for activation makes this current a good candidate [23]. Particularly, it has been proposed that If constitutes the major mechanism increasing the slope of the diastolic depolarisation at low adrenergic tone [226]. This proposal is based on the observation that low doses of the -adrenergic agonist isoproterenol specifically increase the slope of the diastolic depolarisation without affecting the action potential waveform [23]. However, this interpretation assumes that the only ionic current which controls the slope of the diastolic depolarisation is If. The specific role of Cav1.3 channels in the generation of the diastolic depolarisation in mouse SAN cells could challenge this view. Indeed, the strong sensitivity of ICa,L to PKA-dependent phosphorylation, suggest that this current can constitute an important mechanism for converting the status of autonomic input into a change in the heart rate. However, the role of ICa,L in the -adrenergic regulation of pacemaker activity has not been elucidated and will need deep investigation using genetically-modified mouse strains. The role of TTX-sensitive and TTX-resistant INa in the sympathetic regulation of pacemaking is also of great interest. Indeed, cardiac TTX-resistant INa is sensitive to phosphorylation by PKA and PKC [227, 228]. We can speculate that sympathetic stimulation of TTX-resistant INa can accelerate pacemaking by facilitating impulse conduction within the node as well as from the node to the atrium. The effects of autonomic agonists on TTX-sensitive INa are more difficult to predict. Indeed, it has been suggested that these channels could be negatively regulated by the sympathetic nervous system as in neurons [221]. Consequently, the importance of INa during -adrenergic stimulation of pacemaking can exceed that observed under basal conditions, and will be investigated in the future. Recent studies have highlighted the functional association between INa/Ca carried by NCX and RyRs-mediated Ca2+ release in the -adrenergic stimulation of automaticity in pacemaker cells of amphibians [229] and mammals [24, 230, 231]. It has been shown that in the presence of ryanodine, adrenergic receptor activation fails to increase diastolic subsarcolemmal Ca2+ release and, in spite of an apparently preserved stimulation of ICa,L, the positive chronotropic effect on pacemaking is strongly reduced [24]. In another study using SAN intact tissue, ryanodine also significantly reduced -adrenergic stimulation of pacemaking by about 40% [230]. Remarkably, pacemaking in the intact SAN tissue shows less sensitivity to ryanodine than in isolated pacemaker cells. This difference can be explained by the reduced expression of proteins regulating Ca2+ handling in the center versus the periphery of the SAN [39], a difference which is reflected at the functional level by a reduced dependency of pacemaking in the center from SR reticulum Ca2+ release [40]. It has also been shown that ryanodine does not prevent stimulation of pacemaking in isolated SAN cells by cAMP analogs. This effect has been ascribed to stimulation of f-channels gating by cAMP [225]. The joint dependence of pacemaking from proper ICa,L activity [103], SR subsarcolemmal Ca2+ release


[24] and gating of If channels by cAMP [225] is strongly suggestive of a concerted ionic mechanism in the -adrenergic regulation of pacemaker activity in vivo. The interplay between SR Ca2+ release, ICa,L and If in the -adrenergic receptor mediated stimulation of pacemaking will need proper investigation in the future. Ionic Channels and Parasympathetic Regulation of Pacemaking Release of acetylcholine (ACh) from parasympathetic nerve endings reduces the level of intracellular cAMP leading to de-phosphorylation of ICa,L and inhibition of Ist. Also, by reducing the production of cAMP, activation of the muscarinic receptor shifts If activation to more negative voltages [69, 232]. Beside down regulation of cAMP, ACh provokes the opening of KACh-channels [233]. The effects of parasympathetic input on pacemaking are more prominent when SAN rate has been previously elevated by sympathetic tone, a phenomenon named “accentuated antagonism” by Levy [234], to define the dynamical interaction between sympathetic and parasympathetic input to the heart. Synthesis of nitric oxide (NO) following activation of the muscarinic receptor appears to be a major mechanism involved in accentuated antagonism in the dog SAN in vivo [235]. At the cellular level, NO stimulates the activity of guanylyl cyclase, thereby promoting phosphodiesterase II–mediated cAMP breakdown and inhibition of ICa,L [236, 237]. In the SAN, the synthesis of NO constitutes an obligatory mechanism in the muscarinic regulation of ICa,L [238]. Consistently, mice lacking the endothelial NO synthase (NOS) lack muscarinic regulation of ICa,L [239]. Disruption of Go protein by gene inactivation in the mouse also abolishes muscarinic regulation of ICa,L [240]. In conclusion, SAN ICa,L is an important target for both the stimulatory cAMP-dependent intracellular signalling pathway as well for the inhibitory NOS-dependent production of cGMP. This property may be highly significant for coupling ICa,L to different G-protein coupled receptors that can influence heart rate. When applied in isolated SAN pacemaker cells ACh provokes strong negative chronotropic effect by slowing the diastolic depolarisation and hyperpolarizing the maximum diastolic potential. Strong activation of IKACh in vitro can arrest pacemaking [168]. It is a consistent observation that low doses of ACh reduce the slope of the diastolic depolarisation without any significant hyperpolarization of the cell maximum diastolic potential [168]. Also, the same concentrations of ACh have been shown to inhibit If in the absence of activation of IKACh [168]. The relative contribution of ICa,L in the regulation of pacemaker activity has also been studied as the responsiveness of this current to applied ACh compared to IKACh and If [241]. Inhibition of If is observed in rabbit SAN cells for much lower doses of ACh than that required to inhibit ICa,L [241]. This has led to the proposal that the only ionic current controlling pacemaking at low vagal tone is If [168, 226]. Recent evidence obtained in isolated beating mouse hearts indicate that the negative chronotropic effect induced by low doses of ACh is insensitive to block of

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IKACh to the bee toxin tertiapin [242]. This observation seems to support the view that If can sustain regulation of heart rate for weak activation of the muscarinic receptor. Nevertheless, it is not known how these observations from in vitro cellular and organ preparations apply to heart rate regulation in vivo. To this respect, mice inactivated for Kir3.4 and lacking IKACh have strongly reduced autonomic regulation of heart rate in the high and low frequency spectrum of the heart rate variability (HRV) [176]. In the SAN, the muscarinic regulation of ICa,L depends from the level of cAMP, since both basal inhibition of PKA or cell dialysis with a non hydrolysable cAMP analog abolish the responsiveness of ICa,L to ACh [102]. The sensitivity of Cav1.3 channels to ACh has not been yet investigated. To this respect, the relatively positive holding potentials employed thus far to study the regulation of ICa,L (about -30 mV) by autonomic agonists [241] would inactivate most of Cav1.3 channels [19]. In conclusion, it is possible that the proper cholinergic control of heart rate in vivo requires the combined intervention of IKACh as well as the other cAMP-sensitive mechanisms, including If, ICa,L and RyR-dependent diastolic release of Ca2+. Numerical Models of Pacemaker Activity During the last thirty years, several numerical models have been developed to reproduce the initiation and regulation of pacemaker activity. Throughout this period, numerical models have been representative of the best available knowledge of the cardiac pacemaker mechanism. Earlier model versions were structured by classical Hodgkin-Huxley equations for ionic currents and differential equations for calculating the intracellular ionic balance during pacemaking [12, 243-245]. These “first generation” models have been incessantly implemented by new experimental results and finally originated new models that have been developed to take into account the intracellular Ca2+ buffering [246], the autonomic regulation of pacemaking [247, 248], the regional heterogeneity of SAN tissue [13] and the consequences on pacemaking of SR Ca2+ release [15, 249]. Furthermore, models reproducing the activity of the SAN in toto are in progress and aim to integrate large scale three-dimensional numerical simulations of the electrical activity of the whole heart [250, 251]. Here, it is important to stress that the choice and usage of a particular model depends on the scope of the user. Indeed, numerical modelling can be used to predict the effects of manipulating a particular ionic current on pacemaking, especially when no specific pharmacological tools can be employed experimentally. Predictions on the impact of Ist on the diastolic depolarisation rate [186] and the consequences of voltage- and Ca2+-dependent inactivation of ICa,L on pacemaking [99] are just two examples of such an approach. Also, numerical simulations of pacemaker activity can be employed to predict the overall effect of a particular drug on pacemaking when more than one channel is affected, or when different channels are affected at increasing drug concentrations. Finally, numerical modelling can be very effective for interpreting the effects of mutations carried by ionic channels on the cardiac action potential or pacemaker activity [252]. We can expect that models of automaticity will help future research for the development of new drugs



targeting cardiac automaticity [253]. Numerical simulation of pacemaker activity will also be important for understanding the relationship between the genotype and phenotype in inherited diseases of cardiac ionic channels. AN INTEGRATED VIEW OF CARDIAC PACEMAKING AT THE CELLULAR LEVEL Cellular Organisation of the Pacemaker Mechanism From the above discussion, it is clear that the proper generation and regulation of pacemaker activity requires the intervention of multiple classes of ionic channels. Specific ionic channels can act in different physiological situations or can assume a variable relevance depending on the autonomic input or the regional organisation of pacemaker tissue. Also, differences in the density of ionic currents between species exist. Most of these differences can be easily explained by the need to adapt the basal heart rate to the animal size. The species-dependent differences in SAN ionic currents have been reviewed in detail [254]. Fig. (2) summarizes the current knowledge on the different ionic channels and intracellular regulatory pathways involved in automaticity. It is important to stress that, beside ionic channels, pacemaking also depends from the activity of the Na+/K+ pump and NCX. The Na+/K+ pump generates an important current in pacemaker cells which participates in the setting of the maximum diastolic potential in the range of -60 mV [255]. Na+ entry through ionic channels such as If and INa activates the Na+/K+ pump which tends to hyperpolarize the membrane potential [256]. As a consequence, at the end of the action potential repolarization the maximum diastolic potential will be set by the equilibrium between the decaying IKr, the activating If and the Na+/K+ pump. NCX Fig. (2) has been indicated as an obligatory mechanism for maintaining pacemaking. Indeed, increasing the Ca2+ buffering in spontaneously beating amphibian pacemaker cells progressively stops beating in association with decaying of INa/Ca [257]. Abolition of pacemaker activity upon block of NCX activity has also been observed in SAN cells of guineapig [258] and rabbit [259]. In rabbit SAN pacemaker cells, NCX is functionally coupled to local subsarcolemmal Ca2+ signalling [259] (see Fig. 2). Particularly, Ca2+ release from the SR has been proposed to generate an inward current during pacemaking by stimulating NCX activity. There is substantial evidence showing that subsarcolemmal SR Ca2+ release is intrinsically rhythmic and independent from the membrane voltage [217]. SR Ca2+ release could participate to triggering of the diastolic depolarisation even if its contribution is probably more pronounced under activation of the -adrenergic receptor [24, 231]. HCN channels underlie the If current. If is the only ionic current activated in hyperpolarization. Thus, If has the required biophysical properties to constitute the first voltagedependent trigger of the diastolic depolarisation at the end of the action potential repolarization. f-Channels represented in Fig. (2) are densely grouped into “hot spots” that can be isolated in inside-out patches from SAN cells [70]. Further-

more, the existence of membrane lipid rafts concentrating fchannels have been recently demonstrated [260]. It is tempting to speculate that the regulation of f-channels by cAMP actually takes place into a subcellular space delimited by lipid rafts [260] Fig. (2). Given that Na+ influx through fchannels would stimulated the Na+/K + pump, it would be interesting to know if these proteins are also associated in lipid rafts of SAN cell membranes. As discussed above, there exist still uncertainties as to the composition of native fchannels in the SAN. The cardiac phenotype of mice lacking HCN4 and HCN2 channels [20] indicate that these channels participate to the formation of native SAN If. HCN1 channels are significantly expressed in the SAN at the mRNA level [50]. The functional role of HCN1 channels in the generation of native If remains to be established. Cav1.3 channels have been shown to contribute to the diastolic depolarisation [19]. Contribution of T-type channels has also been proposed and is supported by evidence on mice lacking Cav3.1 channels [180]. It remains to be established if VDCCs play an independent role (e.g. by pure inward current flowing) or share the same intracellular Ca2+ regulatory space as RyRs. This attractive hypothesis is suggested in Fig. (2), on the basis of several observations coming from myocardial cells [98]. The definition of the exact nature of the neuronal Na+ channel subunit(s) contributing to automaticity in the adult SAN will deserve further investigation. Indeed, at the mRNA level very low expression of Nav1.1 has been found in the adult mouse SAN, as compared to Nav1.4 channels [50]. On the other hand, mouse SAN cells and tissue sections are positively stained by a specific antibody against the Nav1.1 protein [134]. Future experiments will help elucidate this discrepancy. In Fig. (2), the main ionic current supporting membrane repolarization is IKr. As discussed above, IKr is necessary to drive the repolarization in adult mouse primary pacemaker cells [261] and even partial block of this current drastically affects pacemaking [150]. The involvement of IKr in the autonomic regulation of pacemaking still needs to be elucidated. Biological Pacemakers and Cell Therapy of the Heartbeat: Considerations and Implications for the Mechanism of Automaticity The identification of the ionic channels involved in cardiac automaticity harbours the attractive perspective to engineer “biological” pacemakers to stimulate automaticity in defined regions of the heart. Biological pacemakers have been proposed as a possible future alternative to electronic devices [27, 187]. The first proposal for creating “ad hoc” pacemaker activity came from experiments of adenoviral gene transfer of a dominant negative Kir2.1 subunit (Kir2. 1AAA) [187]. By down regulating IK1 in the guinea pig left ventricle, this construct was able to induce localized pacemaker activity and generated premature ventricular beats in vivo [187]. These experiments have also been presented as a possible challenge of the view that pacemaker activity de-


pends upon the expression of particular ionic channel genes such as HCN. According to this hypothesis, the whole myocardium would be apt to generate automaticity, IK1 expression constituting an endogenous repressor of automaticity outside the cardiac conduction system (CCS) [187]. Even if numerical simulations have suggested that the diastolic depolarisation could be sustained by INa/Ca in ventricular cells transfected with the Kir2.1AAA subunit [262], automaticity in these cells probably lacks the sensitivity to autonomic regulation needed to sustain the everyday life. Creation of biological pacemakers by overexpressing HCN channel subunits is now entering a phase of intensive research [27]. This approach is based on the observation that overexpression of HCN2 or HCN4 channels in cultured neonatal ventricular myocytes strongly enhances automaticity [263, 264]. Consistently with the view that If initiates pacemaking in these myocytes, suppression of HCN channel expression by a dominant negative HCN2 subunit abolished automaticity [264]. It has been shown that adenoviral gene transfer of HCN2 channels in vivo in the canine left atrium induces expression of If in atrial myocytes and is able to generate viable atrial rhythms when SAN activity has been suppressed by vagal nerve stimulation [265]. This strategy has been now improved by specific gene transfer in the CCS, rather than in the myocardial tissue. Adenoviral gene transfer in the left bundle branches of HCN2 channels seems to improve the rate of escape rhythms under vagal suppression of SAN activity [266]. An alternative approach for creating biological pacemakers consists in overexpressing HCN channels in unexcitable human mesenchymal stem cells. These cells have the natural ability to establish gap junctions with cardiac myocytes in culture. Inward current generated in stem cells can drive contractions of myocytes. For a detailed discussion on the clinical and experimental problems associated with stem cells transfection and implantation in the heart, the reader is referred to the review by M. Rosen and co-workers [27]. The possibility to generate biological pacemakers in situ constitutes a fascinating perspective for the cellular therapy of the heartbeat. It remains to be established whether biological pacemakers will necessitate the co-transfection of more than one type of ionic channel in order to match the need for proper autonomic regulation of automaticity. PACEMAKER MECHANISMS AND HUMAN ARRHYTHMIAS In humans, idiopathic or acquired alterations in cardiac pacemaker activity have been associated to alterations of four different ionic channels involved in pacemaking. More generally, dysfunction of cardiac ionic channels [151] or calsequestrin [267] can lead to desynchronization of the sequential activation of the heart and arrhythmias. Here we will focus our attention on dysfunctions of ionic channels affecting pacemaker activity in humans. Sinus node dysfunction (SND) is the major clinical cause necessitating assisted cardiac activation and accounts for approximately half of the number of patients requiring the implantation of an

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artificial pacemaker [268]. The sick sinus syndrome (SSS) is characterised by a combination of symptoms such as dizziness, fatigue, and syncope. SSS can display several electrocardiographic features including sinus bradycardia, sinus arrest, sino-atrial exit block, or alternating periods of bradycardia and tachyarrhythmias [269]. In most cases, this disease state is due to an idiopathic degeneration of the sinus node or is secondary to pharmacologic agents such as those which have antiarrhythmic properties. However, in a significant portion of patients, SND appears in the absence of any identifiable cardiac structural abnormalities or other associated conditions, and displays familial legacy [270-272]. Three mutations have been found to affect the human HCN4 gene and cause congenital sinus node dysfunction in patients. The first of these mutations generates a truncation at the Cterminal part of the gene (573X) and provokes loss of the exercise-induced increase of heart rate [273]. Also a missense mutation in HCN4 (D553N) has been identified [274]. This mutation alters trafficking of HCN4 channels and causes syncope, prolongation of the QT interval as well as polymorphic ventricular tachycardia [274]. The third mutation is associated with familial asymptomatic bradycardia [275]. Affected HCN4 channels are normally responsive to cAMP, but activate for more negative voltages than wildtype channels [275]. Also, L- and T –type channels can be candidates for future research on inheritedSND. Indeed, sinus bradycardia has been recently found to be associated with congenital heart block (CHB) [276]. CHB is a conduction abnormality characterized by complete atrioventricular block that is detected at or before birth in a structurally normal heart. CHB is associated with production of auto-antibodies reactive with the intracellular soluble ribonucleoproteins 48-kDa SSB/La, 52kDa SSA/Ro and 60-kDa SSA/Ro [276]. This study has established that IgGs from mothers with CHB children inhibit ICa,L and ICa,T, indicating that these channels constitute the ionic mechanism by which maternal antibodies induce sinus bradycardia in CHB [277]. Another gene linked to SND is the Nav1.5. Benson and co-workers reported Nav1.5 mutations leading to a recessive disorder of cardiac conduction characterized by bradycardia progressing to atrial unexcitability during the first decade of life [278]. However, it is uncertain if bradycardia observed in patients carrying mutations in the Nav1.5 gene is due to dysfunction of the impulse generation in the SAN. Indeed, another plausible mechanism to explain inherited Nav1.5linked disorders is impaired conduction between the SAN and the adjacent atrial myocardium. Such a mechanism would be consistent with the suggested role for Nav1.5 channels in intranodal conduction in the mouse [134]. As discussed in the preceding sections, mice lacking Cav1.3 [18] and HCN2 channels [192] display sinus dysrhythmia. It is thus tempting to speculate that defects in HCN2 and Cav1.3 channel functions may lead to alterations in pacemaker activity in humans and explain the molecular basis of inherited SND.



PHARMACOLOGICAL CONTROL OF HEART AUTO-MATICITY: PACEMAKER CHANNELS AS THERAPEUTICAL TARGETS It has been known for many years that the mean resting heart rate is associated with all-cause cardiovascular mortality and morbidity. Indeed, association between elevated heart rhythm and all cause mortality has been established by large scale epidemiological studies including the Framingham study [279]. The mechanistic link between heart rate and mortality is not yet known, but on the long term point of view, evidence have been presented supporting the hypothesis that elevated heart rate contributes to the progression of atherosclerosis [280, 281]. Short term effects of elevated heart rate are linked to an augmented risk of cardiac ischemia due to an imbalance between oxygen supply to myocardial muscle and the actual physiological demand. This is particularly true in patients with angina pectoris or coronary disease. Since heart rate is inversely correlated with the degree of filling of the left ventricle with oxygen rich blood, a better irrigation of the coronary system is accomplished with a reduced heart rate. It has been demonstrated that slowing of the heart rate improves myocardial oxygen supply and prevents episodes of angina pectoris [282]. Specific reduction of heart rate has thus been proposed as a new therapeutically effective approach to manage cardiac ischemia [283]. The development of therapeutically active molecules able to reduce the heart rate by prolonging the diastolic time is thus of extreme interest. Pharmacological targeting of ionic channels specifically involved in the regulation of the diastolic depolarisation rate constitutes the natural approach toward this goal. Blockers of the If current have been developed in the past, but the only molecule to have reach the clinical development is ivabradine. Ivabradine is a new heart rate reducing agent, developed for the treatment of stable angina pectoris [25]. In animal models, ivabradine limits exercise-induced tachycardia in the absence of any inotropic [284] or dromotropic effects [285]. Ivabradine administered at pharmacological doses to experimental animals does not affect the QT interval [286]. As expected from a pure effect on the diastolic phase of the cardiac cycle, the consequence of heart rate reduction is an improved balance between myocardial oxygen demand and supply [287]. A phase clinical II study conducted in patients with stable angina treated with ivabradine has confirmed specific heart rate reduction at rest and during exercise and has consistently shown anti-ischemic and anti-anginal efficacy of the drug [288]. Ivabradine induces negative chronotropism in pacemaker cell, by decreasing the rate of diastolic depolarization without affecting the maximal diastolic potential [289]. Electrophysiological recordings on isolated pacemaker cells using the patch-clamp technique have shown a selective use- and current-dependent blockade of If [290, 291]. Also, for concentrations close to that of If half-inhibition, no effect was observed on ICa,L, ICa,T, and IKr, indicating a specific action of ivabradine on fchannels [290]. The in vivo pharmacological properties of ivabradine are consistent with a specific action on If in the

SAN. f-Channels are the first ionic channels underlying pacemaking to be targeted by a therapeutically active drug. It is possible that other channel classes, such as Cav1.3 and Cav3.1 channels will constitute future targets for the development of new molecules which specifically regulate heart rate in the absence of concomitant negative inotropism. CONCLUDING REMARKS Different questions on how the heart rhythm is generated in the SAN and is controlled under physiological and physiopathological conditions remain unanswered. Particularly, it is not know which ionic channels are essential for generating the diastolic depolarisation and which ionic mechanisms play a dominant role in the autonomic regulation of automaticity. Nevertheless, many ionic channels specifically involved in the generation of cardiac pacemaking have been identified. The physiological interactions between these channels during the cardiac pacemaker cycle will be intensively investigated in the near future. Furthermore, “pacemaker” channels are now becoming subject of new drug conception. To this respect, development of specific heart rate reducing agents stress the importance of getting new insight on the functioning of the basic cardiac pacemaker mechanism. ACKNOWLEDGMENTS We thank Elodie Kupfer, Anne Cohen-Solal, Pierre Fontanaud, and Patrick Atger for their excellent technical assistance. Our laboratory is supported by the CNRS, the INSERM, the Action Concertée Incitative in Physiology and Developmental Biology of the French Ministry of Education and Research, the INSERM National Program for Cardiovascular Diseases and the Fondation de France. We also thank the International Research Institute Sevier (IRIS) for having supported the research activity in our laboratory. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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