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Page 1Articles of 56 in PresS. Am J Physiol Heart Circ Physiol (September 7, 2007). doi:10.1152/ajpheart.00750.2007 Electrophysiology and pacemaker function of the developing sinoatrial node Mirko Baruscotti1 and Richard B. Robinson2 1 Laboratory of Molecular Physiology and Neurobiology, Department of Biomolecular Sciences and Biotechnology, University of Milano, Italy and 2 Department of Pharmacology and the Center for Molecular Therapeutics, Columbia University, New York, New York 10032, USA Running title: Electrophysiology of the developing SAN Corresponding author: Richard B. Robinson Department of Pharmacology Columbia University, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-8371 Fax: 212-305-8780 E-mail: [email protected]. 1 Copyright Information Copyright © 2007 by the American Physiological Society. Page 2 of 56 Abstract The sinoatrial node performs its task as a cardiac impulse generator throughout the life of the organism, but this important function is not a constant. Rather, there are significant developmental changes in expression and function of ion channels and other cellular elements, which lead to a postnatal slowing of heart rate and may be crucial to the reliable functioning of the node during maturation. In this review we provide an overview of current knowledge regarding these changes, with the main focus placed on maturation of the ion channel expression profile. Studies on the sodium and pacemaker currents have shown that their contribution to automaticity is greater in the newborn than in the adult, but this age-dependent decrease is at least partially opposed by an increased contribution of the L-type calcium current. Whereas information regarding age-dependent changes in other transmembrane currents within the sinoatrial node are lacking, there are data on other relevant parameters. These include an increase in the nodal content of fibroblasts and in the area of non-expression of connexin 43, considered a molecular marker of nodal tissue. Although much remains to be done before a comprehensive view of the developmental biology of the node is available, important evidence in support of a molecular interpretation of developmental slowing of intrinsic sinoatrial rate is beginning to emerge. Keywords: SAN, cardiac pacemaker, development, Na current, Ca current 2 Copyright Information Page 3 of 56 Introduction The anatomical identification of the sinoauricular junction (now named sino-atrial node, SAN) was originally reported in 1907 by two physiologists/anatomists, Keith and Flack (58). This finding, tog4ether with the earlier identification of other structures of the cardiac conduction system (104, 110, 123), prompted further studies which soon yielded the concept that the heartbeat in mammals originates in the SAN (70, 124) and then spreads to the rest of the cardiac tissue in a temporally and anatomically orderly manner. The impact on the cardiac scientific community was immediate and led to continuing effort to unravel the working mechanisms of this biological impulse generator. As a result, automaticity mechanisms in adult SAN have been extensively studied and have been the subject of numerous reviews (21, 31, 48, 98, 99, 120). Despite this, the basis of automaticity, and the issue of the relative importance of different currents to pacemaking, remains controversial. However, a few generalities can be stated. Pacemaking will depend on current flow during diastole, as well as the presence and characteristics of the major depolarizing currents that take the cell to and beyond threshold. Multiple currents are likely to contribute to both diastolic depolarization and threshold, and the mix varies with species, with disease and regionally within the node. In addition to the characteristics of the channels at the cellular level, pacemaking also will be influenced by characteristics of the tissue, including connections to surrounding atrial myocytes and fibroblasts. 3 Copyright Information Page 4 of 56 Despite the extensive literature devoted to the ionic basis of automaticity in the adult SAN, and the fact that age-dependent differences in intrinsic heart rate have been recognized for some time (52, 53, 87, 114, 127, 128), there has been far less research on the basis of automaticity in the newborn or immature node. There is, however, sufficient evidence to state categorically that the ionic basis of automaticity in the newborn is distinct from that of the adult. These differences can have important implications for the diagnosis and treatment of cardiac rhythm disorders in the immature, adult and aged individual. In this review we will focus specifically on the young versus adult SAN, but will include data on the aged heart where appropriate. We will not describe the adult characteristics in detail, but will refer to published reviews and simply highlight key aspects for the purpose of comparison to the young node. Our intent is, in part, to educate the reader to the fact that the basis of SAN automaticity is quantitatively and qualitatively distinct in the newborn and adult. However, our additional purpose is to highlight how much remains to be determined in this field and thereby to encourage additional research on the topic. General properties of the SAN In mammals the pacemaker region of the heart (the SAN) is located in the wall of the right atrium, but its precise location can vary slightly in different species. In rabbits, a long standing model for heart automaticity studies, it extends along its major axis between the ostii of the superior and inferior venae cavae and along its minor axis between the interatrial septum and the crista terminalis (33), while in human, murine and 4 Copyright Information Page 5 of 56 canine hearts the pacemaker cells are more concentrated in the proximity of the sinus node artery at the junction of the superior vena cava and the right atrium (12, 94, 99). Studies on the cytological organization of the SAN have revealed a structure composed of a dense array of connective tissue in which a small number of specialized cells, all electrically connected, are sparsely, but not homogeneously embedded. Although the SAN as a whole is normally recognized as a uniform functional pacemaker unit, only a small portion of it consists of primary pacemaker cells which are the early generators of the excitatory depolarizing waves that first spread to the rest of the nodal tissue and then to the rest of the myocardium. Indeed several studies at the single cell level have verified that the properties of SAN cells are not identical throughout the entire node, but present noticeable differences that are relevant to their specific role in the context of generation, modulation, and early spread of the impulse (14, 118). For example isolated central nodal cells have a slower rate of contraction and are normally more depolarized than peripheral or secondary pacemaker cells. Furthermore, autonomic stimulation induces a shift in the position of the leading pacemaker, thus revealing an inherent lack of homogeneity within the central nodal region itself (88). The above observations suggest that understanding of intrinsic SAN function must proceed along two lines: one is the elucidation of the ionic and modulatory properties of a single pacemaker cell, and the other is the electrical interaction between nodal cells and nodal-atrial cells. A detailed analysis of the ionic mechanisms generating automaticity requires studies at the single cell level, but to fully understand the complexity of the SAN physiology it is important to consider that each cell exists as an element of a complex network and this network in turn strongly influences the functional characteristics of its component cells (13, 54, 133). For example 5 Copyright Information Page 6 of 56 several papers (13, 55) have shown that central nodal cells must be adequately protected from the more hyperpolarized surrounding atrial tissue since the electrotonic influence of the atrium can profoundly influence SAN activity. This protection, on the other hand, must not be so extreme as to isolate the SAN cells, or the consequence would be that spontaneous activity would be confined within the node, and fail to propagate to the rest of the heart. Molecular markers of the SAN Until recently the only way to precisely identify the SAN region was electrical mapping (26, 126). This technique continues to be utilized in basic and clinical research since it can be applied to both in vivo and in vitro preparations and permits a precise description of the progression of the depolarizing wave. More recently molecular-based identifications have been suggested, based on comparative studies of adult sinoatrial and atrial myocytes that rely on the presence or absence of specific molecular markers. Combined efforts of several groups have determined that adult SAN cells express connexin 45 (Cx45) and neurofilament M (NF-M), but do not express connexin 43 (Cx43) and Atrial Natriuretic Peptide (ANP). This expression profile is completely reversed in atrial cells. Further, crucial differences also exist at the level of ionic currents; for example SAN cells are characterized by the pronounced expression of the pacemaker current, If (see the section below on ionic currents in the developing node), while they express little or no inwardly rectifying IK1 current (24, 32, 48, 85). These data have been largely collected in adult preparations; therefore an important question that should be 6 Copyright Information Page 7 of 56 asked is whether the presence or absence of these molecular markers is stable during the entire lifetime of the organism. If not, this would raise the possibility that variations in the expression of these proteins could be linked to selective and specific age-dependent functional differences in the node which could eventually lead to the occurrence of agingrelated pathologies. Further, a strong developmental regulation of any of these proteins would impact their utility as markers in the young or perhaps aged SAN. Expression of Neurofilament M Studies have explored the expression of NF-M in the conduction system of the adult heart, as well as during embryonic development in rabbits (39, 121). Positive staining for NF-M is considered an appropriate hallmark for the identification of this specialized tissue (33, 35). NF-M can be considered an early marker of the developing conduction system of the embryo since it is already detectable at E9.5 just before heart looping has completed (121), and its expression profile is complete by birth (40). However, in considering the relevance of NF-M as a tool to identify and investigate the properties of the pacemaker region, it is important to note that there may be marked species differences, since there has been no evidence for the presence of NF-M in humans (121) and rats (5). Expression of connexin isoforms Connexins can serve both as molecular markers and as important contributors to the electrophysiological properties of the node. Connexins are proteins that when properly assembled give rise to gap-junctions that allow the intercellular exchange of ions and 7 Copyright Information Page 8 of 56 small molecules. In the human and mouse genome the family of connexins includes at least 20 members (106). For several years the isoforms known to be expressed in cardiac tissue were Cx40, Cx43, and Cx45. This list has been recently updated by Kreuzberg et al. (64) who also reported the presence of Cx30.2. The distribution of these isoforms is not uniform throughout all heart regions. Indeed Cx40 and Cx43 are mostly present in the working atrial and ventricular myocytes, while conduction tissue is characterized by the presence of Cx45 and Cx30.2 (the mouse homolog of human Cx31.9). In particular, Cx30.2 seems to be selectively expressed in the SA and atrioventricular (AV) nodes (64). This pattern of expression has been particularly useful in studies of the SAN since the absence of Cx43, originally observed in several studies (28, 29, 86, 112, 116) has been utilized to identify the SAN region from the surrounding Cx43-rich atrial tissue. In terms of functionality, Cx45 has a somewhat lower unitary conductance than either Cx40 or Cx43 (17, 18). Further, Cx30.2 has the lowest unitary conductance of all gap junctional proteins, and knocking out this connexin results in a 25% decrease in the PQ interval (63). Thus, the nodal specific connexin expression pattern may be critical to its property of slow conduction. FIGURE 1 NEAR HERE Jones et al. (52) have investigated the expression profile of Cx43 in the SAN throughout the lifetime of the guinea-pig. They found Cx43 to be uniformly present throughout the 8 Copyright Information Page 9 of 56 node in newborn animals, but progression from youth to senescence was accompanied by a 14-fold increase in the nodal area lacking Cx43 and a corresponding decrease in the level of Cx43 protein expression (Fig 1). Interestingly, and in agreement with previous data, the disappearance of the Cx43 initiated in the central nodal area and then extended to more peripheral nodal regions with further aging. Furthermore there was no increase in Cx40 and Cx45 to compensate for the loss of Cx43. Two key questions regarding this example of developmental regulation of Cx43 are (1) what is its influence on functional aspects of the pacemaker site (i.e. does the disappearance of Cx43 relate to the intrinsic heart rate and/or nodal propagation)? (2) What are the modulatory signals that control Cx43 expression? While the latter is still unanswered, some work has been done regarding the former. In particular Yamamoto et al. (126) reported that the nodal area in which Cx43 is absent is characterized by a 5-fold decrease in conduction velocity, and this is consistent with the concept that a dependence solely on Cx45 and Cx30.2 would lead to slower conduction. Furthermore Jones et al. (52) observed that the distance that the impulse generated at the center of the node must travel before reaching atrial tissue (identified by the crista terminalis) increases with age. Taken together these findings provide a molecular correlation to the functional observation of a decrease in the overall intrinsic heart rate and conduction time with age. Fibroblast content of the developing node The fibroblast component is one of many factors differentiating SAN structure from that of other cardiac regions. For example, in humans the fibroblast content of the adult working myocardium is about 5-6% of the total volume, but this proportion is much 9 Copyright Information Page 10 of 56 higher in the SAN, where it reaches values of 45%-75% (19, 27, 103). Studies on humans and lower mammals also have highlighted that the fibroblast, and therefore collagen, content of the heart is subject to strong developmental regulation: it is low during initial embryogenesis, and increases during late embryogenesis and postnatal growth (3, 19, 27, 103). After this period of expansion, the proliferation halts but can be triggered by the occurrence of pathological states. The apparent lack of a symmetrical organization of the myocellular component of the node is also true for the fibroblasts since they are found both adjacent to (and thus in contact with) sinoatrial myocytes and as isolated groups (20). It is interesting to note that these two separate populations also display different types of connexins. Fibroblasts interacting with nodal cells preferentially express Cx45, while those that do not contact SAN cells express Cx40; Cx43 has not been found in sinoatrial node fibroblasts (20). The contribution of the fibroblast population to the physiology of the node is complex, but important roles certainly include mechanical support and regulation of local inflammation (105). Since fibroblasts are non-excitable, their dense presence in the SAN, and the observation that they are at least partially coupled via functional gap junctions to nodal cells, pose the intriguing question of a possible electrical consequence. In this regard an interesting observation is that fibroblast resting potential is influenced by stretch as a direct consequence of the presence of stretch-activated ion channels on their cell membrane (56, 60). Whether changes in the membrane potential of fibroblasts directly influence the chronotropism of coupled nodal cells is an interesting question that awaits investigation. 10 Copyright Information Page 11 of 56 In addition to the observations above concerning fibroblast distribution during early development, changes in the structural properties of the SAN associated with aging result in slow conduction and reduced voltage amplitude on eletroanatomic mapping (59, 95). Interestingly these functional changes display some similarities with those observed in pathological states such as sinoatrial node dysfunction (SND) (43, 59, 95). Finally, besides their above mentioned roles, it is possible that fibroblasts contribute to the metabolic homeostasis of the node (19). Ionic channels in the developing node Pacemaker (If) current Pacemaker cells lack a stable resting potential, instead exhibiting a slowly rising depolarization that encompasses the interval from the end of action potential repolarization to the upstroke of the subsequent action potential. The events underlying this diastolic depolarization are the main determinants of cardiac pacemaker rate. Since its early discovery If has been identified as a contributor to diastolic depolarization (15), and indeed its activation upon hyperpolarization and its inward and time-dependent Na+/K+ mixed nature are well-suited to drive cell depolarization during the diastolic interval. The vast majority of information on the biophysical and molecular properties of this current derives from studies in single SAN myocytes isolated from adult animals (6, 25, 31, 93, 108). In addition to its contribution to basal pacemaking, If is a preferential target in the autonomic modulation of cardiac chronotropism due to the presence of a cAMP binding domain on the channel’s C-terminus. The channels responsible for If (f 11 Copyright Information Page 12 of 56 channels) are modulated by both the sympathetic and parasympathetic branches of the autonomic nervous system; activation of the muscarinic cascade affects f channels by reducing the cAMP bound to the channel, thereby inducing a hyperpolarizing shift of the activation curve, while the stimulation of K-adrenoceptors elicits an increase in cAMP available for binding to the channel and thus increases f current during diastole due to a depolarizing shift of the activation curve. To assess whether the pacemaker current undergoes developmental changes that could at least partially explain the slower heart rate observed in adult animals Accili et al. (2) carried out comparative studies of If in SAN myocytes isolated from newborn (9-10 day old) and adult rabbits. They found that newborn SAN cells had both a greater If density (newborn: 0.244 pS/pF, adult: 0.158 pS/pF), and a significantly steeper slope of the activation curve (slope factor: newborn, 9.6 mV; adult, -11.3 mV) when compared to adult cells, but no difference in the midpoint of activation (newborn, -66.7 mV; adult, -66.3 mV). These results, in addition to the observation that the capacitance of SAN cells does not vary with development (7), indicate that the amount of If that flows at pacemaker potentials is greater in newborn animals than in adults and thus could contribute to the higher intrinsic heart rate of rabbit newborn SAN (114). The intrinsic variation of SAN rate during development has also been studied by Yang et al. (127, 128). These authors carried out radioimmunoassay experiments to evaluate agedependent changes in the basal concentration of cAMP in sinoatrial cells and reported a marked decrease (0.31 LM at 2 weeks, vs 0.025 Lm at 12 weeks). They also reported a negative shift of the If threshold potential (M45.5 vs M51.1 mV in 2 week vs 4 week hearts, respectively). Based on these findings the authors proposed that heart rate slowing 12 Copyright Information Page 13 of 56 during development can be partially attributed to a reduced contribution of the pacemaker current secondary to a decrease in cellular cAMP content. The observation that cAMP levels change during development is interesting, but any extrapolation from these data must be done with caution since there is increasing evidence that membrane proteins such as ion channels exist in highly controlled microdomains where the concentration of cytoplasmic molecules such as cAMP can be different from those of the bulk cytoplasm. Furthermore the data of Yang et al. (127, 128) differ from those of Accili et al. (2), who reported no differences in either the midpoint or the acetylcholine- and isoproterenolinduced shifts of the If activation curves between newborn and adult animal. Although Accili et al. (2) did not observe an age-dependent change in the cAMP responsiveness of f channels, this does not preclude an age-dependent difference in the modulation of the current or heart rate. The developmental differences in the biophysical properties of the channels (higher conductance and steeper activation relation in the newborn) means that an identical shift in the position of the activation curve would per se lead to a greater current variation and thus to a more marked effect of autonomic control of heart rate in the newborn, as indeed has been observed both for vagal (30) and K-adrenergic (44) stimulation. This is illustrated in Fig 2, where the activation parameters of Accili et al. (2) have been used to plot the relative activation relations of newborn and adult SAN cells under basal conditions and under the influence of high concentrations of acetylcholine and isoproterenol, to represent maximal vagal and sympathetic tone, respectively. As shown, at a potential of -55 mV, which is the approximate maximum diastolic potential in the SAN, the amount of current contributed by If under maximal vagal tone is approximately the same in newborn and adult. However, with maximal 13 Copyright Information Page 14 of 56 sympathetic tone the current increases only 2.8-fold in adult, compared to 4.5-fold in newborn, representing a 60% greater change in current at the younger age. FIGURE 2 NEAR HERE Molecular identification of pacemaker channels, either at the message or protein level, has been carried out in several species, including rabbits, mice, rats, and dogs (38, 46, 49, 71, 72, 73, 96, 97, 100, 102, 115, 135). There are convincing indications that in the adult SAN each functional channel is composed of 4 subunits belonging to the family of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels. The major component of the native SAN pacemaker current is generally the HCN4 isoform (49, 71, 79, 81, 102), although a recent study using laser capture and RT-PCR unexpectedly found that in rat SAN the major isoform at the message level is HCN2 (46). While these data on transcript expression need to be extended to the protein level, this same study also reported changes in message as a function of age. Both HCN2 and HCN4 transcript expression progressively decreased with development, and consistent with this observation, the non-selective f-channel blocker cesium became less effective in slowing rate with development (46). To our knowledge this is the only study of pacemaker currents in the developing rat SAN, and so the results await confirmation by other groups and extension to other species. 14 Copyright Information Page 15 of 56 The presence of HCN4 signal is generally considered a marker for the early identification of the SAN region during embryonic development (80). Although when singularly expressed in heterologous system all members of the HCN family display similar properties, the most distinguishing characteristic of HCN4 is its slow kinetics (typical time constants of activation at -95 mV are: 0.11, 1.13, and 2.52 s for HCN1, HCN2 and HCN4, respectively) (1, 4). These kinetics can be modified by specific interactions with physiological modulators such as PIP2 or MiRP1 (MinK Related Protein 1), which profoundly increase the current density and speed up the activation process of some HCN isoforms (89, 92, 131). Although to our knowledge no specific data are available on the molecular identification of the newborn f currents in rabbit heart, the observation by Accili et al. (2) that its properties are similar to those of adult animals would tend to suggest a similar molecular assembly. Further evidence in support of a prevalent HCN4 composition of newborn pacemaker channels comes from embryological data in mice. In a pivotal study Stieber et al. (107) generated both global and cardiac-specific HCN4 knock-out mice and showed that these mice die at about day E9.5-E11.5 due to severe functional anomalies of the cardiac conduction system. Analysis carried out in this period confirmed that mice lacking HCN4 were characterized by a severe bradycardia (88 bpm for HCN4-/- vs 139 bpm for wt HCN4+/+) likely caused by a 75%-90% reduction in the If current. Interestingly, cardiac rate of knockout mice could not be modulated by a cAMPdependent mechanism, thus strengthening the evidence that cAMP-dependent rate modulation is mostly induced by an If-dependent mechanism. Given the molecular composition and biophysical properties of the pacemaker currents during embryological and post-natal development, it is reasonable to conclude that HCN4 subunits are central 15 Copyright Information Page 16 of 56 elements of cardiac pacemaker myocytes at all ages. Thus, an important aspect of future studies will be the analysis of the genetic mechanisms that regionally and temporally control or repress HCN4 channel expression. In this respect, Kuratomi et al. (65) recently have investigated some aspects of the molecular mechanism controlling expression of HCN4 and have identified both the promoter region responsible for basal activity and the presence within the first intron of a conserved Neuron-Restrictive Silencer Element (NRSE) sequence. NRSE is a sequence that when bound to the Neuron-Restrictive Silencing Factor (NRSF), inhibits the transcriptional processing of neural genes in non neuronal cells. Interestingly, Kuratomi et al. (65), have found that the NRSE-NRSF system plays a crucial role in controlling the known progressive decrease of HCN4 channels during embryological development of the working myocardium (129). Indeed, the levels of NRSF signal increases during embryological development thus leading to a substantial inhibition of the transcriptional activity of the HCN4 gene. It is worth noting that the same mechanism operating in the opposite direction controls the increased expression of HCN4 channels induced by cardiac hypertrophy (36, 65, 66). Finally, the role of the NRSF-NRSE system in controlling channel expression is not limited to HCN4, but is also believed to control transcription of T-type calcium channels (66, 130). The extent to which these regulatory mechanisms are active in the developing sinoatrial node remains to be determined. Information on the mechanism controlling the differential fate determination of cardiac embryonic cells bound to become sinoatrial node or atrial cells are also currently becoming available. As shown by Hoogaars et al., (45) the transcriptional repressor Tbx3 seems to be the key element inducing the formation of the SAN from the rest of the 16 Copyright Information Page 17 of 56 (Tbx3-free) future working atrial myocardium. Whether or not the presence of Tbx3 is directly involved in the expression of HCN4 channels is not known. However, it is likely that the elucidation of the molecular events controlling expression of the pacemaker phenotype will have practical implications for understanding arrhythmogenic cardiac diseases and innovative therapeutic strategies. Sodium currents Until a decade ago it was commonly accepted that there was little or no functional contribution of Na current to the activity of primary sinoatrial pacemaker cells. This conclusion was mostly derived from the observation that the Na channel blocker tetrodotoxin (TTX) did not affect the spontaneous rate of either the intact SAN (61, 62, 69, 125) or single cells from adult rabbits (82, 83). In more recent years the view that Na current is irrelevant to pacemaker activity has been profoundly modified by developmental studies in the rabbit and by studies of adult SAN in other species (7, 24, 34, 68, 75). FIGURE 3 NEAR HERE In 1996 we reported that a Na current was present in rabbit primary nodal cells at birth. Its developmental regulation was evident from the observation that by the 40th postnatal day the current had fully disappeared (7). The functional role of the current was assessed 17 Copyright Information Page 18 of 56 by challenging the spontaneous activity of newborn cells with the Na channel blocker TTX (3 LM). Somewhat unexpectedly, TTX modified all action potential parameters rather than only the upstroke velocity. Particularly intriguing was the decrease in the slope of the diastolic depolarization (from 0.035 to 0.015 V/s) and the consequent slowing of spontaneous rate from 90 to 33 beats per minute. Similar investigations of adult SAN cells failed to demonstrate INa and, as expected, TTX did not affect rate. This marked age-dependent difference in the effect of TTX on spontaneous rate is illustrated in Fig 3. Whole-cell and single channel kinetic analysis demonstrated that a slow inactivation process due to multiple single channel re-openings at pacemaker potentials was the cause for the small but significant inward current that, in addition to If, contributed to diastolic depolarization (8, 9). The contribution of this current to diastolic depolarization is best seen under ramp clamp conditions, where the membrane potential is slowly depolarized and the net current recorded. When TTX is used to block the contribution of any Na current, one can determine the TTX-difference current. As shown in Fig 4 (panels A and B), there is a marked TTX-sensitive inward current during this membrane depolarization in a newborn, but not an adult SAN cell. A further characterization of this developmentally regulated newborn SAN Na channel unexpectedly revealed a pharmacological profile (i.e. high TTX, and low cadmium sensitivity) that did not correspond to that of the cardiac Na channel isoform (Nav1.5), but was rather more typical of a neuronal isoform. The neuronal character of the Na channel in the newborn SAN was confirmed by in situ hybridization experiments that revealed the expression of Nav1.1 in the newborn SAN (10). This explained the 18 Copyright Information Page 19 of 56 functional contribution of the channel, since inactivation of neuronal Na channel isoforms typically requires greater depolarization than the cardiac isoform. Thus Na current derived from neuronal channel isoforms would have greater availability at the typical diastolic potentials of the SAN. The results with TTX also made it apparent that the Na current could not provide the only difference between newborn and adult pacemaker activity. As evident in Fig 3, the Naindependent automaticity of newborn cells unveiled by the use of TTX (33 bpm) was significantly slower than that of adult cells (68 bpm). Based on these results it appears that the presence of a Na current during early postnatal development is a consequence of the immature state of other sinoatrial currents, which alone are not sufficient to maintain a cardiac rate compatible with metabolic requirements. This critical situation resolves during the first 40 days of life in the rabbit, thus removing the necessity for the current. It is noteworthy that while most currents contributing to pacemaker activity in the adult such as If, Ca current and Na/Ca exchanger are highly modulated by autonomic agonists, with cAMP dependent processes enhancing the currents, (11, 16, 119, 132) this is less true for INa. Modulation of Na channels is isoform specific, and protein kinase A can be associated with either an increase or decrease in current (22, 122). For this reason understanding of the functional contribution of Na channel modulation to the control of pacemaker mechanisms is still unclear. However, when taken together these observations lead to the highly speculative hypothesis that during the early postnatal period the contribution of INa ensures a failsafe mechanism protecting the animal from a potential 19 Copyright Information Page 20 of 56 parasympathetic/sympathetic imbalance, and that the necessity for this fades during development as the autonomic nervous system reaches its mature state. Investigation of the presence of Nav1.1 and Nav1.5 transcripts in the adult rabbit by Tellez et al. (111) found Nav1.5 in the periphery and low levels of Nav1.1 in the center, compatible with the almost negligible contribution of INa to adult automaticity as previously suggested by Baruscotti et al. (7). Interpretation of these data together with those obtained in newborn rabbit could suggest that the expression of Na channel in the center of the node is regulated differently than the expression in the periphery. In fact, it would be expected that Nav1.5 channels could only participate in peripheral regions due to the more negative inactivation relation of this isoform, and thus are more involved in the propagation of the impulse out of the node rather than to determination of rate. These conclusions were further validated by experiments in mice having a targeted disruption of the SCN5A (Scn5a+/-) gene. Peripheral SAN cells isolated from these animals displayed a reduced presence of Na currents and reduced spontaneous rate; furthermore experiments on intact SAN preparations confirmed that in Scn5a+/- mice there was a significant slowing of SA conduction time and a propensity to sino-atrial block (67). To our knowledge, thorough comparative studies of SAN Na currents in newborn and adult have not been carried out in any other species, and thus the progressive disappearance of Na currents during development could be a process typical of some, but not all species. Indeed, studies carried out in adult mice show significant differences with those from rabbits. Recordings from single SAN cells revealed that pacemaking rate in 20 Copyright Information Page 21 of 56 adult mice is extremely sensitive to TTX, and in fact Na currents were recorded in almost all cells investigated (68, 75). In 2004 Lei et al. (68) concluded that although Na currents are common throughout the whole node of adult mice, their molecular substrates are not the same in all regions. Cardiac Nav1.5 channels are absent from the center of the SAN, but densely populate the periphery, while Nav1.1 are homogeneously distributed throughout the node. It is possible that the persistence of Nav1.1 expression in the adult mouse SAN, compared to the rabbit, reflects the need to maintain a markedly higher heart rate in the adult mouse heart. While the mechanism(s) controlling the differential regulation of Nav1.1 and Nav1.5 are unknown, a report (101) of developmentally regulated splice variants of the murine SCN5a gene with distinct cardiac specific enhancer regions, and the presence of homologous human splicing, suggests a direction for future research. Calcium currents Two types of calcium currents are expressed in SAN cells: the low voltage activated (ICaL) and the high voltage-activated (ICaT) (42, 117). The relative abundance of these two currents seems to be species-specific since, for example, in rabbits ICaL is the predominant current while in mice the opposite situation occurs. Although several details still need to be fully understood there is general agreement that L-type calcium current sustains the action potential upstroke (phase 0), either alone or in combination with Na current, and contributes to the later part of diastolic depolarization (76, 117, 132). The specific role of ICaT is more controversial since original electrophysiological studies on pacemaker cells were carried out in rabbits, a species in which the T-current is poorly 21 Copyright Information Page 22 of 56 expressed. With the more recent focus on pacemaking mechanisms in mice, studies have revealed that in this species T-current participates in diastolic depolarization (78). A further role for ICaT has been associated with its ability to locally trigger Ca2+ release from the sarcoplasmic reticulum in atrial cells (47), although this specific action has not yet been proved in primary pacemaker cells (77). FIGURE 4 NEAR HERE Given their importance, the obvious question is whether calcium currents are subjected to developmental regulation in the SAN. As mentioned earlier, when the contribution of Na currents in rabbit newborn cells is removed by TTX, the remaining automaticity is slower than that of the adult (33 vs 68 bpm, (7)). A logical explanation for this observation is that other ionic components crucial for nodal automaticity are still not fully mature in the newborn, and the calcium currents are obvious candidates. Protas et al. (91) carried out a detailed investigation of developmental changes in nodal ICaL and ICaT. Comparative analysis of ICaT in newborn and adult rabbit SAN cells did not show any difference, thus excluding it as a major player in the developmental regulation of SAN rate in the rabbit. Taken at face value the absence of differences in newborn and adult T-type currents, in spite of the differences in rate in the presence of TTX, might indicate that in rabbits ICaT is not a critical determinant of rate. However, the only conclusion that can be definitively drawn from these data is that, to the extent that ICaT contributes to automaticity in the 22 Copyright Information Page 23 of 56 rabbit SAN, it is a developmental constant. Again, this may reflect species differences since it has been shown that spontaneous rate of Cav3.1 (one of 3 T-type isoforms) knockout mice is significantly slower (~10%) than that of control animals (78). In contrast, when Cav3.2 knockout mice were investigated for chronotropic performance and atrioventricular conduction of the impulse, no alterations were observed (23). Interestingly Niwa et al. (84) have shown that in the mouse heart Cav3.2 expression is high during embryogenesis. Opposite to Cav3.2, Cav3.1 increases during late embryogenesis and at birth to become the only expressed isoform found in the heart after birth and during adulthood (37, 84). These molecular data are consistent with the observation by Protas et al. (91) that in the rabbit the properties of newborn and adult sinoatrial ICaT current are not different. More interesting are the results comparing L-type Ca currents in adult and newborn (91). In particular, development acts on ICaL currents by inducing a decrease in current density (newborn, 17.6 pA/pF; adult, 12.1 pA/pF), a rightward shift of the inactivation curve (midpoint: newborn, -33.4 mV; adult, -28.3 mV), and a leftward shift of the activation curve (midpoint: newborn, -17.3 mV; adult, -22.3 mV). Recovery from inactivation was also slower in the newborn. The decreased density of the current observed in the adult would suggest a diminished contribution of this current during diastole, but the opposite is likely true when one considers that the overlapping area delimited by the activation/inactivation curves is increased in the adult due to the opposite shift of the curves. This is illustrated in Fig 4 (panels C and D), where the nifedipine sensitive current is shown during a slow ramp clamp to mimic diastolic 23 Copyright Information Page 24 of 56 depolarization. While the use of nifedipine during a ramp clamp can isolate currents in addition to ICaL, such as the sustained inward current (see below and (24, 41)), it is evident that the threshold of the nifedipine sensitive current is more negative in the adult, so that it contributes earlier in diastole. Mean data confirm this, with the measured threshold shifted 4 mV negative in the adult (n=6-8; P<0.05). To the extent that this reflects L-type current it is thus likely that even though maximal current is higher in the newborn, the more negative activation threshold, and the faster recovery from inactivation observed in the adult could explain why in the presence of TTX the rate of newborn cells is slower than that of adult. The mechanisms underlying the differences observed in newborn and adult ICaL could arise from a developmental switch between channel isoforms. Four R subunits of the Ltype calcium channels have been identified and those expressed in the heart are Cav1.2 and Cav1.3, with the latter being mainly expressed in the atrium, in the SAN and conduction system (12, 76, 79, 109). When Cav1.3 knockout mice were generated SAN dysfunction and disturbances of AV conduction were observed (76, 90, 134). Analysis of sinoatrial ICaL current in Cav1.3 knockout mice revealed significant differences with those recorded in wild type; in particular in the absence of Cav1.3 the voltage dependence is shifted ~ 5 mV positive (midpoint from -16.6 to -11.4 mV in (134)) and this shift demonstrates that in the SAN two different isoforms of L-type calcium channels coexist. In particular since Cav1.3 is selectively present in supraventricular regions, and since cardiac rate is slower when this isoform is absent, it is possible that Cav1.3 in the SAN mostly contributes to diastolic depolarization, while contractility is contributed to 24 Copyright Information Page 25 of 56 by the Cav1.2 isoform. Interestingly when the derivative of the action potential was measured at three different potentials (-50, -45 and -40 mV) during diastolic depolarization in wild type and Cav1.3 deficient mice, differences were observed only for the two more positive voltages (134). These data indicate that the contribution of Cav1.3 is indeed critical during the pacemaker phase, but not in early diastole where other currents contribute. Whether Cav1.2 and Cav1.3 are subjected to developmental regulation remains to be established. Based on the 5 mV developmental leftward shift in activation observed by Protas et al. (91) and the 4 mV shift in threshold of the nifedipine sensitive current reported above, when considered in comparison to the 5 mV shift in the activation relation in the Cav1.3 knockout (134), it could be speculated that the immature SAN expresses a prevalence of Cav1.2 channels, and postnatal development favors the expression of Cav1.3. This hypothesis also agrees with the evidence that while Cav1.3 is not necessary during heart embryogenesis, the lack of Cav1.2 is lethal. Additional support for this hypothesis comes from the finding of Jones et al. (51) of a progressive loss of Cav1.2 with aging in the guinea pig SAN, however it was not reported if a concomitant increase in Cav1.3 occurred. Other currents Other currents have been suggested to contribute to SAN pacemaking in the adult by various researchers, including the deactivation of the delayed rectifier current IK (48), a nifedipine-sensitive sustained cation current called, Ist (24, 41) and Na/Ca exchange current (74). We are not aware of any studies in the immature sinoatrial node that indicate any of these currents vary developmentally, but this is clearly a relevant question. In 25 Copyright Information Page 26 of 56 particular the Na/Ca exchange mechanism merits study, as this is intimately associated with other components of Ca homeostasis such as sarcoplasmic reticular Ca stores and release mechanisms, both of which change with postnatal development in other cardiac regions (50, 57, 113). Conclusions We have attempted to provide a framework for considering the developmental maturation of the SAN in mammals. While any conclusions must be qualified because of the possibility of substantive differences among species, it appears the SAN undergoes substantial rearrangements both in its anatomical extension, as shown by the decrease in the Cx43 area, and in its electrical aspects, as shown by profound modifications of the ionic channel expression profile. All these aspects combine to determine an agedependent slowing of intrinsic heart rate and conduction time. Interestingly we have observed that while the contribution of If and INa to pacemaking are down-regulated during postnatal life in the rabbit SAN, the opposite is true for ICaL. Certainly these events are controlled by an overall homeostatic developmental plan, but identification of the common regulators is a goal that remains for future investigations, as is extension to other species including human. What can be stated at this time is that although some aspects are beginning to be clarified, much remains to be done before a comprehensive view of the developmental biology of SAN function will be complete. 26 Copyright Information Page 27 of 56 Acknowledgments Supported by NIH HL-28958. We thank Drs. Dario DiFrancesco and Michael R. Rosen for helpful comments on the manuscript. 27 Copyright Information Page 28 of 56 References 1. 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Functional Roles of Ca(v)1.3 (alpha(1D)) calcium channel in sinoatrial nodes: insight gained using gene-targeted null mutant mice. Circ Res 90: 981-987, 2002. 135. Zicha S, Fernandez-Velasco M, Lonardo G, L'Heureux N, Nattel S. Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model. Cardiovasc Res 66: 472-481, 2005. 50 Copyright Information Page 51 of 56 Figure legends Figure 1: Sinoatrial node development and aging are accompanied by a progressive reduction of Connexin 43 Distribution (top) and expression (bottom) of the junctional protein connexin 43 in the sinoatrial node of Duncan-Hartley guinea pigs of different ages (<1 day up to 38 months) were evaluated by immunofluorescence analysis. The area lacking Cx43 signal, calculated from a two-dimensional reconstruction of the whole SAN obtained from sequential slices, increased linearly with the age of the animal. The quantification of the expression of connexin 43 was accomplished by western-blot and densitometric analysis and normalized to desmin expression, which is not influenced by development or aging. Data are reproduced, with permission, from panels 3B and 4B of (52). Figure 2: The range of If current availability under autonomic control is greater in the newborn than in the adult. Activation curves of the pacemaker current obtained in control (continuous line) and in the presence of maximal sympathetic (isoproterenol) or vagal (acetylcholine) activation. The range of fractional activation at -55 mV, a voltage near the maximal diastolic 51 Copyright Information Page 52 of 56 potential, is 60% greater in the newborn than in the adult. The parameters of the activation curves were taken from Accili et al., 1997 (2). Figure 3: Contribution of INa to spontaneous activity decreases with post-natal development. Action potentials recorded in single cells isolated from an adult and a newborn rabbit sinoatrial node exhibit a different sensitivity to the Na channel blocker TTX (3 LM). Drug perfusion induces no significant effect (-10.6 % beats per minute, n=5, p>0.05) on spontaneous rate in the adult animal, while it profoundly (-66.7 % bpm, n=5) alters rate in the newborn. (modified from (7)). Figure 4: TTX- and nifedipine-sensitive currents elicited by ramp depolarization differ in newborn and adult SAN cells. A, B: Whole-cell current traces obtained in control and in the presence of TTX (3 LM) by applying a voltage ramp (from -60 mV to -20 mV) to single SAN cells at a depolarizing rate (35 ms/s) similar to that of spontaneous diastolic depolarization (top). TTX-sensitive current (A, B bottom) is present in the newborn, but not in the adult. C, D: Similar protocols allow the identification of nifedipine-sensitive currents (bottom) both in the newborn and in the adult. On average, the threshold of the nifedipine-sensitive current is 4 mV less negative in the newborn. 52 Copyright Information Cx43 protein expression (%) Area absent of Cx43 protein expression (mm2) Page 53 of 56 50 40 30 20 10 0 Neonate Young Adult Old Senescent 70 60 50 40 30 20 10 0 Neonate Young Adult Age of animal Copyright Information Old Senescent Normalized Activation Copyright Information 0.0 0.5 1.0 1.5 -120 2.8x -80 -60 Voltage (mV) -100 ADULT Normalized Activation (Relative to Adult) -40 0.0 0.5 1.0 1.5 -120 -80 -60 Voltage (mV) -100 4.5x NEWBORN -40 Page 54 of 56 Voltage (mV) Voltage (mV) Copyright Information -60 -40 -20 0 20 40 -60 -40 -20 0 20 40 Control NEWBORN 0.5 Control ADULT 1.0 Time (s) TTX TTX 1.5 2.0 Page 55 of 56 Current (pA/pF) Current (pA/pF) Current (pA/pF) Copyright Information Current (pA/pF) -20 -1.5 -1.0 -0.5 0.0 0.5 -1.5 TTX Nifedipine-sensitive 0.5 0.0 -0.5 -1.0 -1.5 -2.0 -2.0 0.0 -1.0 4.0 6.0 2.0 +Nifedipine -1.5 -1.0 -0.5 0.0 0.5 -1.5 -1.0 -0.5 -0.5 0.5 -2.0 -60 0.0 C -30 TTX-sensitive -40 Voltage (mV) 0.0 0.5 -2.0 -60 0.0 Control 4.0 6.0 -1.5 -50 +TTX 2.0 A -1.0 -0.5 0.0 NEWBORN D B -50 +TTX Control TTX -20 Nifedipine-sensitive +Nifedipine TTX-sensitive -40 -30 Voltage (mV) ADULT Page 56 of 56