© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
The sinoatrial node, a heterogeneous pacemaker structure
aSchool of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK
bResearch Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan
* Corresponding author. Tel.: +44-113-233-4298; fax: +44-113-233-4224 m.r.boyett{at}leeds.ac.uk
Received 23 November 1999; accepted 18 May 2000
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Abstract |
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 Top Abstract 1 Introduction2 Anatomy of the...3 Electrical coupling in...4 The functioning of...5 Extracellular potentials from...6 Regional differences as...7 Pacemaker shift8 Electrical heterogeneity of...9 Computer modelling10 Autonomic regulation11 Deterioration in the...12 ConclusionsReferences |
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This article focuses on the regional heterogeneity of the mammalian sinoatrial (SA) node in terms of cell morphology, pacemaker activity, action potential configuration and conduction, densities of ionic currents (iNa, iCa,L, ito, iK,r, iK,s and if), expression of gap junction proteins (Cx40, Cx43 and Cx45), autonomic regulation, and ageing. Experimental studies on the single SA node cell to the whole animal are reviewed. The heterogeneity is considered in terms of the gradient model of the SA node, in which there is gradual change in the intrinsic properties of SA node cells from periphery to centre, and the alternative mosaic model, in which there is a variable mix of atrial and SA node cells from periphery to centre. The heterogeneity is important for the dependable functioning of the SA node as the pacemaker for the heart, because (i) via multiple mechanisms, it allows the SA node to drive the surrounding atrial muscle without being suppressed electrotonically; (ii) via an action potential duration gradient and a conduction block zone, it promotes antegrade propagation of excitation from the SA node to the right atrium and prevents reentry of excitation; and (iii) via pacemaker shift, it allows pacemaking to continue under diverse pathophysiological circumstances.
KEYWORDS Gap junctions; Ion channels; Sinus node
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1 Introduction |
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TopAbstract1 Introduction2 Anatomy of the...3 Electrical coupling in...4 The functioning of...5 Extracellular potentials from...6 Regional differences as...7 Pacemaker shift8 Electrical heterogeneity of...9 Computer modelling10 Autonomic regulation11 Deterioration in the...12 ConclusionsReferences |
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The sinoatrial (SA) node is the pacemaker of the heart. More than any other tissue in the heart (apart from the atrioventricular node) the SA node is a complex tissue and its function depends on this complexity. This is the central thesis that this review will attempt to establish. From the study of the electrophysiology of single SA node cells, in particular, the pacemaking of the SA node is reasonably well understood. However, the relatively simple scenario that has emerged from these studies cannot account for the undoubted complexity of the SA node — for such well known phenomena as the marked heterogeneity of electrical activity throughout the SA node, the non-radial spread of the action potential from the leading pacemaker site in the SA node, the block of conduction from the leading pacemaker site towards the atrial septum, and pacemaker shift. We will attempt to show that these phenomena are related to the problems of (i) the relatively small SA node having to drive the large mass of atrial muscle without it being suppressed by the more hyperpolarized atrial muscle, (ii) protecting the SA node from reentry and invasion from action potentials from outside the SA node, and (iii) how pacemaking in the SA node must continue under diverse circumstances.
There have been many books and reviews on the SA node [1–9]. The first comprehensive monograph on SA node function and morphology was published by Brooks and Lu in 1972 [1]. One review includes the interesting history of research into the SA node [4], one focuses more on the SA node of the dog and human [3] and another focuses on the ionic currents of the SA node [5].
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2 Anatomy of the SA node |
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TopAbstract1 Introduction2 Anatomy of the...3 Electrical coupling in...4 The functioning of...5 Extracellular potentials from...6 Regional differences as...7 Pacemaker shift8 Electrical heterogeneity of...9 Computer modelling10 Autonomic regulation11 Deterioration in the...12 ConclusionsReferences |
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2.1 Gross architecture
Fig. 1 shows a schematic diagram of a SA node–atrial muscle preparation from the rabbit [14]. In all species, the SA node is located in the right atrium at the junction of the crista terminalis (a thick band of atrial muscle at the border of the atrial appendage) with venous tissue — the superior and inferior vena cava, and the intercaval region between the two great veins [4,10–16]. The size of the SA node varies in different species and is reviewed by Opthof [4,17]. In the human, published photographs show the SA node beneath the epicardial surface of the crista terminalis [18,19]. In other species, at least part of the SA node lies in the intercaval region. In the cat, the SA node from the intercaval region can rise up the epicardial face of the crista terminalis before terminating [14]. In the dog, the SA node in the intercaval region appears to abut against the crista terminalis [20]. In the rabbit, SA node tissue from the intercaval region rises up the endocardial face of the crista terminalis and terminates at the right branch of the sinoatrial ring bundle (Fig. 1). Similarly, in the monkey SA node tissue rises up the endocardial face of the crista terminalis [16]. In most species (rabbit, rat, guinea-pig, cat, dog, pig, monkey [12–16,20–22]), perhaps even human [19], but apparently not cow [21], the SA node may extend from the superior to the inferior vena cava.
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Within the intercaval region, the SA node may occupy the entire thickness between the endocardium and epicardium as in the rabbit, guinea-pig and monkey [12,13,16]. However, in the human, dog and pig there is a layer of atrial muscle between the SA node and endocardium [15,18–20]. The atrial muscle in the intercaval region together with extensive connective tissue (see below) is thought to protect the region against the high wall stresses.
One question to be considered in this review is how the relatively small SA node can drive the large mass of atrial muscle surrounding it without being suppressed by it. Theoretically this is difficult [23]. It is possible that this is in part achieved by restricting the connections between the SA node and atrial muscle, because contact between the two tissues over a large area would result in greater suppression of the SA node pacemaker activity. For this reason it is of interest to know how the SA node is connected to the atrial muscle; for example, is the SA node electrically connected to the atrial muscle throughout their common boundary? It is possible that for much of the overlap between the SA node and atrial muscle there is connective tissue separating the two. Such a connective tissue barrier has been described in the human and cow [21] (but not in human according to Anderson and Ho [11]), rabbit [24] and monkey [16]. The connective tissue barrier in the rabbit is shown schematically in Fig. 2 as the thick black band.
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2.2 Fine architecture
A characteristic feature of the SA node is much connective tissue, mainly collagen and fibroblasts [25], although the extent of connective tissue is species-dependent and varies from 50% in the rabbit, guinea-pig and rat to 75–90% in the cat [4]. Another characteristic feature of the SA node under the light microscope is a high density of nuclei; in the monkey, the density in the SA node is approximately double that in the atrial muscle [16]. The high density of nuclei reflects the fact that SA node cells are small as compared to the surrounding atrial cells. The cells in the centre of the SA node are reported to be 5–10 µm in diameter in the human and dog [26], roughly spindle-shaped, 25–30 µm long and <8 µm in diameter in the rabbit [12], 20–30 µm long with an irregular profile in cross section and a diameter of <8 µm in the guinea-pig [13], <10 µm in diameter (often only 4–6 µm) in the cat [27], 40 µm long and 4–8 µm in diameter in the pig [15], and irregular and spindle-shaped with a diameter of
7 µm in the monkey [16]. In contrast, atrial cells in these species are 100 µm in length and 15–20 µm in diameter. In the centre of the SA node, the cells are poorly organised and described as ‘interweaving’ in the human, dog and rabbit [12,26]. In the human and dog, in the centre of SA node, there are characteristic ‘P’ cells (or ‘typical nodal’ cells), which are believed to be the leading pacemaker cells [26]. The cells are not only small as reviewed above but also ‘empty’. This is because they contain only a few poorly organised myofilaments (running in all directions and not organised into myofibrils). In the human and dog, typical nodal cells also contain fewer and randomly distributed mitochondria and little sarcoplasmic reticulum [26]. Empty cells are characteristic of the SA node in a number of species: rabbit [12], guinea-pig [13], cat [14], pig [15] and monkey [16]. However, the cells are less empty in the cat, pig and monkey, and they contain more and better organised myofilaments than in the other species [4]. In rabbit [12], guinea-pig [13], cat [14], pig [15] and monkey [16], the presence of numerous caveolae on the cell membrane has been noted. The absence of myofilaments and mitochondria can be understood in a specialised tissue like the SA node, but the presence of numerous caveolae has not been explained. Could they increase the area of membrane available to receive neurotransmitters? An abundance of glycogen granules has been noted in the rabbit [12].
In the rabbit, there is a gradual transition in cell type over several millimetres from the centre of the SA node (the leading pacemaker site) in all directions to the periphery of the SA node, where the SA node meets the atrial muscle; there is no distinct border between the SA node and atrial muscle [12,28]. From the centre to the periphery of the SA node, the shape and arrangement of the cells become more regular and the myofilaments more numerous and better organised. In panels A and B of Fig. 1 the stippled area shows the region of small interweaving SA node cells, the yellow area (without stipples) shows the region of larger transitional SA node cells and the dashed yellow line shows the extent of peripheral SA node tissue lying over the atrial muscle of the crista terminalis. The myofilament content is least in the region of small interweaving cells and gradually increases in all directions from this region [28]. In the human and dog, there is evidence of a similar gradual transition in cell morphology [21,26]. James et al. [26] described the nodal cell as the main cell in the SA node in the human and dog, but in addition to these there are ‘transitional cells’ with an increasing number of myofilaments and mitochondria. All stages of transition can be found, with some cells being similar to typical nodal cells and others similar to atrial cells. Some transitional cells are half nodal cell like and half atrial cell like. The transitional cells are located between the nodal cells and the atrial cells and James et al. [26] proposed that conduction from the nodal cells to the atrial cells occurs via the transitional cells. However, in the cat SA node, Opthof et al. [14] described a sharp transition between nodal cells in the SA node and atrial cells outside. Consistent with the description above of a gradual transition in cell type from the centre to the periphery, cells of various sizes can be isolated from the rabbit SA node [29]. Isolated cells can also have various morphologies: Denyer and Brown [30] and Verheijck et al. [29] classified rabbit SA node cells into elongated spindle, spindle and spider cells — spider cells, unlike elongated spindle and spindle cells, have multiple cytoplasmic projections.
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3 Electrical coupling in the SA node |
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TopAbstract1 Introduction2 Anatomy of the...3 Electrical coupling in...4 The functioning of...5 Extracellular potentials from...6 Regional differences as...7 Pacemaker shift8 Electrical heterogeneity of...9 Computer modelling10 Autonomic regulation11 Deterioration in the...12 ConclusionsReferences |
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3.1 Conduction velocity and space constants
Based on mathematical modelling, Joyner and van Capelle [23] concluded that some degree of electrical uncoupling of the cells within the SA node may be an essential design feature to protect the leading pacemaker site from the atrial muscle. The conduction velocity within the SA node is very low (0.03–0.05 m/s — Section 4.1) compared to that in the surrounding atrial muscle (
1 m/s). Conduction velocity is determined by a variety of factors, but one of the most important is the coupling conductance between cells. The low conduction velocity in the SA node is one line of evidence of poor electrical coupling in the SA node. The space constant (the distance over which an electrotonic potential, caused by intracellular current injection, decays to 1/e of its original value) is a measure of electrical coupling between cells. In the rabbit SA node, it has been measured to be between 465 and 828 µm parallel to the crista terminalis and between 205 and 310 µm perpendicular to it [31–35]. The difference in space constants in the two directions can explain the anisotropy in the SA node, i.e. the higher conduction velocity parallel to the crista terminalis than perpendicular to it (see Section 4.1). The difference in the space constants in the two directions is probably the result of the orientation of cells in the rabbit SA node — the cells tend to be arranged in parallel with the crista terminalis [12,28]. The space constant of SA node is lower than that of atrial muscle [36] and this is again consistent with less electrical coupling in the SA node than in the atrial muscle.
3.2 Connexin phenotypes in the SA node
Ultrastructural studies using the electron microscope have demonstrated the presence of gap junctions in the SA node of rabbit, mouse, rat, bat, mole, dog, sheep, cow, monkey and man, although they are sparse and smaller in size compared with those in the surrounding atrial muscle [37]. Masson-Pévet et al. [38], using electron microscopy, estimated that gap junctions along the cell border at the leading pacemaker site in the rabbit SA node occupy 0.2% of the cell border. From this, assuming a typical SA node cell is 6 µm in diameter and 20 µm in length with a surface area of 1000 µm2, they calculated the total surface area of gap junctions to be 2 µm2. The area of individual gap junctions in the same study was 0.87x10–2 µm2 [38]. Therefore, the number of gap junctions in the cell membrane of one primary pacemaker cell can be estimated to be 230 from the morphological observations.
Each gap junction comprises clusters of serially linked hemichannels (connexons) contributed by the two apposing cell membranes, giving a way for small molecules (<1 kDa) to pass between the two cell interiors [39]. Each connexon is composed of six transmembrane proteins called connexins, a multigene family of conserved proteins, of which at least 13 members are known in mammals [40,41]. In the heart, mRNA for several connexins has been detected: Cx37, Cx40, Cx43, Cx45 and Cx46 [42–44]. Gap junction channels made from different connexin types in vitro are reported to show distinct unitary conductance, ionic selectivity and molecular permeability properties [45,46]. Cx43 is ubiquitous and abundant in the working myocardium (atrial and ventricular muscle) [39,47,48]. This is the not the case in the specialised conducting tissues of the heart [49–51].
Many immunohistochemical studies have focused on connexin phenotypes in the SA node, but the results have been inconsistent and conflicting. Anumonwo et al. [52] described Cx43-containing gap junctions in the rabbit SA node. Trabka-Janik et al. [53] also showed clear labelling of hamster SA node, the location of which had been confirmed by action potential mapping, with Cx43 antibodies. Other studies on rabbit, rat, guinea-pig, dog, cow and human, however, have failed to detect Cx43 in the SA node [21,24,47,50,54–57]. In the rabbit, Coppen et al. [24] found that Cx43 expression in the centre of the SA node is negligible compared to that in the surrounding atrial muscle, and that Cx40 and Cx45 are expressed in the Cx-43-negative area (Fig. 2). The dimensions and quantities of the Cx40 and Cx45 spots observed were much smaller than those of Cx43 spots in the atrial muscle, and this is consistent with the size and frequency of SA node cell gap junctions as revealed by electron microscopy (see above). Verheule et al. [54] also showed the absence of Cx43 in the pacemaker cells in the central part of the rabbit SA node, but they demonstrated the presence of Cx40 and Cx46 gap junctions in those pacemaker cells. Davis et al. [50,56] demonstrated the presence of Cx40 and Cx45 in the dog and human SA node region where Cx43 was undetectable.
Although discrepancies between different studies may be due to species differences in connexin expression in the SA node, the discrepancies may also be due to technical problems. Recognition of a specific immunohistochemical signal may be more difficult in SA node cells than in atrial and ventricular cells, because the gap junctions are smaller. The mean number of channels in each SA node gap junction in rabbits is estimated to be 90 channels [38]. This is close to the limit for detection by immunofluorescence [57]. Therefore, the absence of labelling does not necessarily mean the absence of the connexin. Complex anatomical architecture at the junction of the SA node and atrial muscle (interdigitations) as described below will make the situation unclear; the positive Cx43 labelling reported by some investigators might reflect gap junctions in atrial cells between layers of SA node cells. Unreliability of antibodies (cross reaction with other types of connexin) may also lead to false conclusions [49].
Anumonwo et al. [52] and Verheule et al. [54] measured macroscopic gap junction conductance (Gc) of rabbit SA node cell pairs and obtained variable Gc with average values of 2600 and 7500 pS, respectively. Single gap junction channel conductance was estimated to be 40–60 pS by Anumonwo et al. [52] and 133–241 pS by Verheule et al. [54]. From these values, SA node pacemaker cells were considered to be coupled by only 30–60 functional gap junction channels. This is less than the 230 gap junctions per cell estimated from electron microscopy (see above), but there are several possible reasons for this (the number of gap junction channels per cell will be greater than the number of gap junction channels between any two cells; some gap junction channels may not be functional). Because of the high membrane resistance of SA node cells (1 G
), Anumonwo et al. [52] suggested that the minimal Gc required for the synchronisation of excitation would be low, 140 pS (three gap junction channels). In support of this suggestion, Cai et al. [58] in a modelling study determined that approximately four gap junction channels of 50 pS are needed for frequency entrainment. In experiments using an external circuit that couples two cells that are not physically connected, Verheijck et al. [59] demonstrated Gc-dependent electrical behaviour of coupled rabbit SA node cells: as Gc was progressively increased the cells exhibited (i) independent pacemaking, (ii) complex activity with mutual interactions, (iii) entrainment of action potential frequency at a 1:1 ratio with different action potential waveforms, and (iv) 1:1 frequency entrainment with virtually identical action potential waveforms. The critical value of Gc for 1:1 frequency entrainment was only 130–500 pS. These observations suggest that SA node pacemaker cells can be synchronised with a low Gc provided by a limited number (<10) of gap junction channels.
3.3 Spatial distribution of connexins in and around the SA node
Joyner and van Capelle [23] suggested that a gradual increase of intercellular coupling from the centre of the SA node towards the periphery is important for proper functioning of the SA node: in a computer model, such a change in intercellular coupling allowed the SA node to show pacemaking and drive the surrounding atrial muscle, whereas with an abrupt change in intercellular coupling the SA node was unable to drive the atrium or the SA node was made quiescent by the atrial hyperpolarizing load. In order to shed light on this issue, ten Velde et al. [57] examined the spatial distribution of Cx43 in atrial muscle surrounding and abutting the guinea-pig SA node. They used an immunohistochemical marker (anti-
smooth muscle actin, -SMA) that specifically cross-reacts with guinea-pig SA node cells together with Cx43 antibody to label previously electrophysiologically mapped SA node. There was no gradual increase in the Cx43 labelling density at the border between the SA node and atrial muscle. Instead, there was an intermingling (or interdigitation) of strands of atrial cells (Cx43-positive but -SMA-negative) and SA node cells (Cx43-negative but -SMA-positive) [57]. A similar interdigitating arrangement of bundles of Cx43-negative SA node and Cx43-positive atrial cells at the periphery of the SA node was reported in a immunohistochemical study on rat, cow and human hearts [21]. Such interdigitation of well-coupled atrial cells and poorly coupled SA node cells might be important to ensure that SA node pacemaker cells are shielded from the hyperpolarizing influence of atrial muscle and yet able to drive the atrial muscle.
In the study of Kwong et al. [60], the dog SA node was composed mainly of Cx43-negative cells (many of them Cx40-positive), but amongst these were bundles of Cx43-positive SA node cells (also Cx40- and Cx45-positive). The Cx43-positive bundles appeared to abut atrial cells. In the same study, complementary data were obtained from dissociated cells from the dog SA node — two main populations of pacemaker cells were identified: 30–35% of cells expressed Cx43, Cx40 and Cx45 and 55% of cells expressed only Cx40 (the remaining 10–15% of cells had no detectable connexin expression). In the rabbit SA node, Coppen et al. [24] found that most boundaries between Cx43-positive cells and Cx43-negative (but Cx40- and Cx45-positive) SA node cells were sharply delineated, and no extensive interdigitation between the two cell types was apparent. Instead, in the periphery of the SA node both Cx43 and Cx45 were expressed (yellow region on crista terminalis in Fig. 2).
Cx43 in the dog SA node and in the periphery of the rabbit SA node could serve an important role as the preferential conduction pathway for the propagation of the action potential from the centre of the SA node to the atrial muscle. Such differential spatial expression of gap junction protein might provide the structural substrate for the putative transitional zone proposed by Joyner and van Capelle [23] to enable the SA node to drive the atrial muscle surrounding it and yet not be suppressed by it. More extensive experimental studies using a combination of electrophysiology and immunohistochemistry will be required to substantiate this possibility.
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4 The functioning of the intact SA node |
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TopAbstract1 Introduction2 Anatomy of the...3 Electrical coupling in...4 The functioning of...5 Extracellular potentials from...6 Regional differences as...7 Pacemaker shift8 Electrical heterogeneity of...9 Computer modelling10 Autonomic regulation11 Deterioration in the...12 ConclusionsReferences |
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4.1 Activation sequence and regional differences in the action potential in the rabbit
The leading pacemaker site and the activation sequence in the rabbit SA node are shown in Fig. 1A (activation sequence can be seen as a movie at http://www.leeds.ac.uk/bms/staff/boyett/). In Fig. 1A the isochrones show the time in milliseconds for the action potential to conduct from the leading pacemaker site. The leading pacemaker site (asterisk) is just a small fraction of the total area of the SA node (Fig. 1A). Bleeker et al. [12] estimated that the action potential is first initiated in an area of 0.1 mm2 comprising
5000 cells. This is just 1% of the total area of the SA node [4,12]. The leading pacemaker site is located in the centre of the SA node and is typically 0.5–2 mm from the crista terminalis in the intercaval region in the area of small interweaving cells (Fig. 1A) [12]. From here the action potential propagates preferentially in an oblique cranial direction towards the crista terminalis. Bleeker et al. [12] calculated the conduction velocity to be 2–8 cm/s or less around the leading pacemaker site. In experiments in which extracellular potentials were recorded from the endocardial surface, Yamamoto et al. [61] calculated the conduction velocity near the leading pacemaker to be 4.5 cm/s parallel to the crista terminalis and 3.0 cm/s perpendicular to it. In the periphery of the SA node, conduction was more rapid: the conduction velocity was 49.7 cm/s parallel to the crista terminalis and 36.3 cm/s perpendicular to it [61]. The outcome of this is that the action potential arrives at the crista terminalis as a broad wavefront (Fig. 1A). In relation to the problem of the SA node driving the atrial muscle, the arrival of the action potential at the atrial muscle as a broad wavefront may have advantages, because if the action potential arrived at the atrial muscle at a point, it is possible that the action potential could be more easily suppressed by the atrial muscle. Fig. 1A also shows that conduction towards the interatrial septum is blocked. Activation of the interatrial septum must wait for the action potential to conduct around the top or bottom of the block zone (dark grey area in Fig. 1A). This block zone is thought to be physiologically important — it may have a protective function and help protect the SA node from reentry and invasion by action potentials from outside of the SA node [34].
Like cell morphology (Section 2.2) the form of the action potential is not uniform within the SA node. The diversity of action potentials has been extensively studied in the rabbit SA node [12,28,62–64]. In the centre of the SA node, the upstroke of the action potential is slow (<10 V/s), the action potential overshoot is low (<10 mV), the action potential is long (150 ms), the maximum diastolic potential is low (–60 to –70 mV) and the pacemaker potential is steep. From the centre of the SA node to the periphery of the SA node and then atrial muscle of the crista terminalis, there is a gradual increase in the action potential upstroke velocity and overshoot and maximum diastolic potential and a gradual decrease in action potential duration and steepness of the pacemaker potential. It has already been commented on that there is a progressive increase in myofilament content from the centre to the periphery of the SA node (Section 2.2) and Masson-Pévet et al. [28] showed that in the rabbit there is an inverse correlation between the slope of the pacemaker potential and volume density of myofilaments. In the periphery (but not centre) of the SA node, the action potential can also have a notch [62,63].
The changes in the action potential are complex and occur in two dimensions. The action potential duration is greatest at or near the leading pacemaker site and it decreases in all directions from this as shown in Fig. 3A (position of leading pacemaker site shown by asterisk in Fig. 3A). The distribution of action potential duration is roughly similar to the activation sequence (Fig. 1A). Thus, there is a downward gradient in action potential duration along the conduction pathway in and around the SA node. Because of this marked gradient in action potential duration, repolarization in the SA node occurs in the opposite direction to depolarization in the SA node [61,65] (illustrated by a movie at http://www.leeds.ac.uk/bms/staff/boyett/). A downward gradient in action potential duration along the conduction pathway appears to be a general rule in the heart (other examples being crista terminalis vs. atrial appendage, Purkinje fibres vs. ventricular muscle, ventricular sub-endocardium vs. sub-epicardium and ventricular base vs. apex) and is thought to be a protective mechanism to help prevent reentry [65]. It is interesting that the action potential duration gradient in the SA node is substantially greater than that elsewhere in the heart [65].
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Down the centre of the intercaval region, as well as the maximum for action potential duration (Fig. 3A) there is also a maximum for pacemaker slope (Fig. 3C) and minima for action potential peak, diastolic potential and upstroke velocity (Fig. 3B) [65]. However, the parameters are not distributed in the same way. The leading pacemaker site (asterisk in Fig. 3A–C) occurs in the region of maximum action potential duration as already mentioned (Fig. 3A) and maximum pacemaker slope (Fig. 3C), but not minimum action potential peak, diastolic potential and upstroke velocity (Fig. 3B). In Fig. 3 the block zone is shown by the thick black line — the block zone occurs in the region of minimum action potential peak and upstroke velocity (Fig. 3B), but not maximum action potential duration (Fig. 3A) and pacemaker slope (Fig. 3C).
In the block zone, a spectrum of action potentials can be seen: they are usually small and slow and, although some have a normal appearance, the action potentials often show two components [12,34,34,65,66]. In the block zone in the inferior part of the SA node, depolarizations with amplitudes of only a few tens of millivolts, rather than action potentials, and even stable resting potentials have been observed (Fig. 4) [65]. Interestingly, the resting potentials can be –75 mV [65], whereas the resting potential of the SA node at the leading pacemaker site (when spontaneous activity is stopped) is –40 mV [67]. The block of conduction in this zone could be the result of poor electrical coupling between cells or poor excitability. The results above are suggestive of poor excitability and this was also suggested by the study of Bleeker et al. [34] (see also Opthof et al. [66]). Bleeker et al. [34] showed that the block zone in the rabbit is a region of complete block rather than just slow conduction, because if the action potential was prevented from propagating around the region of block (by cutting the tissue superior and inferior to the block zone) the action potential was seen to fail to propagate across the block zone — it died out. They showed that the two component action potentials often seen in the block zone are the result of the collision of two wavefronts — the one attempting to propagate across the block zone and the one that has travelled around the region of block and is invading the block zone from the other direction. Finally, Bleeker et al. [34] showed that the space constant of the block zone is similar to that elsewhere in the SA node — this suggests that electrical coupling is at least as good as elsewhere in the SA node. Support for this comes from the observation that there is no break in connexin expression in the block zone [24].
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4.2 Activation sequence and regional differences in the action potential in other species
Preferential conduction from the leading pacemaker site in the oblique cranial direction and conduction block towards the septum are also seen in the cat [14] and pig [15]. In the guinea-pig, a similar pattern is seen, although the block zone is differently placed [13]. In the monkey, the activation sequence is different in that the action potential from the leading pacemaker site propagates preferentially in an oblique caudal direction to the crista terminalis [16]. However, the effect is the same — the action potential arrives at the crista terminalis as a broad wavefront. In the monkey, as in the other species, there is conduction block from the leading pacemaker site to the septum [16]. In the guinea-pig [13], cat [14], pig [15] and monkey [16] similar regional differences in the action potential are seen to those in the rabbit, although two component action potentials in the block zone are unusual in the cat [14] and absent in the guinea-pig [13]. In the guinea-pig [13] and pig [15], but not the cat [14], there is an inverse correlation between the slope of the pacemaker potential and volume density of myofilaments in SA node cells [68].
Compared to other species, much less is known of the activation sequence and regional differences in the action potential of the SA node in the dog and human. In the dog, extracellular electrodes on the epicardial surface have been used to map the spread of electrical activity [69]. Similar recordings have been made intraoperatively in patients [70]. In patients, the position of the apparent origin of the action potential measured in this way is highly variable (from close to the superior vena cava to close to the inferior vena cava) and there can even be multiple widely separated simultaneous points of origin. A similar picture is seen in the dog [69]. However, it is unlikely that pacemaking in the dog and human is radically different from that in the small mammals. In both the dog and human, the SA node tissue is embedded in atrial muscle (but presumably electrically isolated from it) (see Section 2.1) and extracellular electrode recording at the tissue surface cannot discriminate between potentials resulting from SA node tissue and potentials resulting from atrial muscle (Section 5). Bromberg et al. [22] recorded the activation sequence from the epicardial surface of dog hearts in the way described and then made intracellular recordings from the SA node (in the adult dog, this is difficult and the epicardium had to be first removed from above the SA node tissue). It was shown that the activation sequence of the SA node tissue (recorded with intracellular microelectrodes) was not correlated with the activation sequence determined by extracellular electrodes [22]. It is likely that the origin (even multiple origins) of the action potential when mapped using extracellular electrodes represents the exit point from the SA node to the atrial muscle and not the leading pacemaker site [22]. The nature of these ‘exit points’ is unknown. The true activation sequence of the SA node of the dog (or human) has not been determined (because of technical difficulties) and presents a future challenge. However, Bromberg et al. [22] and earlier Woods et al. [20] made some intracellular recordings from the dog SA node and from these it can be tentatively concluded that the activation sequence and regional differences in the action potential (e.g. higher upstroke velocity in the periphery of the SA node) are not fundamentally different from those in small mammals. There is only one report of intracellular recordings of action potentials from the adult human SA node [71].
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5 Extracellular potentials from the SA node |
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TopAbstract1 Introduction2 Anatomy of the...3 Electrical coupling in...4 The functioning of...5 Extracellular potentials from...6 Regional differences as...7 Pacemaker shift8 Electrical heterogeneity of...9 Computer modelling10 Autonomic regulation11 Deterioration in the...12 ConclusionsReferences |
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Any difference in transmembrane potential between electrically coupled cells should cause a current to flow through the cell interior, across the cell membrane and through the extracellular fluid (‘volume conductor’), giving rise to a potential gradient in the extracellular fluid.
5.1 Animal experiments
The extracellular potential changes associated with the pacemaker activity of the SA node were first recorded by Cramer et al. [72] from rabbit atria using a unipolar electrode (0.5 mm in diameter) connected to a high-gain direct-coupled amplifier. They found a slow negative wave (slope, –30 to –90 µV/s) and a more rapid negative wave (slope, –400 to –800 µV/s) closely correlated to the pacemaker potential and upstroke of leading pacemaker cells. This was confirmed by Haberl et al. [64] who also found variations of the extracellular potentials dependent on the length of SA node conduction time. Similar extracellular potentials were recorded by Cramer et al. [73] from the dominant pacemaker site of the dog SA node in vitro as well as in vivo with the use of low frequency (0.1 to 50–100 Hz) band pass filters. In all of these early reports, the SA node potentials were interrupted by large high-frequency deflections as cells in the surrounding atrium depolarized. In the SA node region, the amount of volume conductor is small and the electrical coupling between cells is weak compared to those in atrial muscle [52,74]. Extracellular potential changes localised to the SA node are, therefore, easily masked by relatively large far-field potentials from the atrial muscle near the SA node. This inhibits study of the morphology of the extracellular potentials in relation to the spread of excitation in and around the SA node.
Recently, Yamamoto et al. [61] reported a better technique to record extracellular potentials in the SA node. They used modified bipolar electrodes with a tip diameter of 0.1 mm. The indifferent electrode was placed 1 mm above the recording site to minimise far-field potentials by a high level of common-mode rejection. Fig. 5 shows recordings from the endocardial surface of the rabbit SA node. Extracellular potentials showed a variety of morphologies. In a small area near the leading pacemaker site, a slow negative wave was preceded by a gradual increase of the negativity, and was normally followed by a second slow negative wave (Fig. 5B: m). At the periphery of the SA node superior and inferior to the leading pacemaker site, slow positive/negative waves were recorded (Fig. 5B: e, t). On the septal side of the SA node with slow conduction, long, slow, positive waves were recorded (Fig. 5B: f, n). In the atrial muscle surrounding the SA node, the extracellular potentials showed a sharp positive wave followed by a short negative wave (Fig. 5B: j, o, u). When transmembrane action potentials were recorded simultaneously with the leading pacemaker-type extracellular potentials, the initial slow negative wave coincided with the upstroke phase of the action potential, whereas the second slow negative wave coincided with the repolarization phase of the action potential, and the gradual increase of negativity at the end of the electrical diastole corresponded with the terminal phase of the diastolic depolarization. These characteristics are consistent in part with previous reports [64,72,73], but the interruption of the waves by sharp deflections reflecting atrial activity (far-field potentials) was minimal or negligible. This made it possible to interpret a wide variety of morphologies of extracellular potentials in terms of the local current generated at the recording site. For instance, the cells at the leading pacemaker site in the rabbit SA node depolarize first but repolarize last because of a prominent gradient of action potential duration from the centre of the SA node, through the periphery of the SA node to the atrial muscle (Section 4.1). Consequently, the leading pacemaker site may play a role as a current source not only during depolarization but also during repolarization. This may result in the characteristic dual negative waves in the extracellular potentials. Pacemaker shifts induced by vagal nerve stimulation or pharmacological block of the Na+ current, iNa, or the L-type Ca2+ current, iCa,L, were also shown to result in changes in the morphology of the extracellular potentials [61]. These facts suggest that the endocardial extracellular potentials recorded in and around the SA node under appropriate conditions may provide useful information, helping recognition of the leading pacemaker site and alterations of the conduction pattern and excitability of mammalian SA node [75].
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However, care has to be taken in extrapolating the results obtained in rabbits to other species. The rabbit SA node is a superficial structure just beneath the endocardium, whereas the SA node of other species including human can be embedded within atrial tissue (Section 2.1). In these species, the extracellular potentials recorded from the endocardial surface will be the result of conduction from the exit point from the SA node and as a result the earliest extracellular activation may not correspond to the position of the leading pacemaker site as already discussed (Section 4.2).
5.2 Clinical implications
Recording of extracellular potentials from the SA node through catheter electrodes was first introduced into clinical electrophysiology in the 1980s [76–79]. This technique was expected to have an advantage over indirect atrial pacing methods in assessment of conduction disturbances from the SA node to the surrounding atrial muscle [80–82]. Identification of the SA node potential could also be important for preservation of normal pacemaker activity in catheter ablation procedures to treat reentrant atrial tachyarrhythmias. The potential usefulness of the technique has been, however, limited by large far-field potentials from atrial muscle close to the SA node [83,84]. This is probably due to the use of a relatively large catheter electrode tip (diameter >1 mm), and a large distance between the recording site and the indifferent electrode (10–15 mm). A closer setting of the indifferent electrode just above a recording tip of smaller size would result in a better isolation of potentials localised to the SA node as described in Section 5.1. The SA node potential recorded from patients through catheter electrodes is usually monitored in the reversed-polarity to see upward going potentials at the time of depolarization. This convention originally employed by Cramer et al. [72,73] and Haberl et al. [64] may be misleading. Extracellular potential waves are caused by local current through the extracellular fluid in association with a propagation of excitation; an action potential approaching the recording site from upstream produces a positive wave, whereas an action potential going away from the recording site downstream produces a negative wave. Accordingly, an alteration of the activation sequence can produce a fundamental change of the extracellular potentials even though a change of the transmembrane action potential may be minimal [61]. We propose that the extracellular potentials recorded in and around the SA node should be presented using the correct polarity to facilitate the translation of their morphology into two-dimensional propagation patterns of excitation.
Introduction of new technology for more precise topological recognition of recording sites, such as catheter-based electroanatomical mapping using a magnetic field [85,86], may also help to draw more valuable information from the SA node extracellular potentials for understanding the complex pathophysiology of human SA node dysfunction.
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6 Regional differences as studied using small balls |
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TopAbstract1 Introduction2 Anatomy of the...3 Electrical coupling in...4 The functioning of...5 Extracellular potentials from...6 Regional differences as...7 Pacemaker shift8 Electrical heterogeneity of...9 Computer modelling10 Autonomic regulation11 Deterioration in the...12 ConclusionsReferences |
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In 1982 we developed the technique of isolating a strand of small ball-like tissue specimens from the centre to the periphery of the rabbit SA node [87]. This is an extension of a technique for the preparation of small balls of SA node tissue for double-microelectrode voltage clamp originally employed by Noma and Irisawa in 1976 [88]. Fig. 6A shows a diagram of a preparation from the rabbit that included the whole SA node from which six balls were made from the sites indicated by the filled circles. Fig. 6B compares the characteristics of the action potentials from the six balls (closed circles) with those of action potentials recorded from the same tissue before the dissection (open circles); all of the data are plotted as a function of the distance from the sinoatrial ring bundle (the border between the SA node and atrial muscle). The two sets of data are similar and this suggests that the small balls are viable and show similar properties to tissue in the intact SA node. An important corollary of this observation is that the continuous gradient of action potential characteristics from the centre to the periphery of the SA node (Fig. 3) is the result of regional differences in the tissue and not the result of electrotonic interaction between the SA node and the surrounding atrial muscle.
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6.1 Spontaneous action potentials
In balls from the periphery, the action potential upstroke velocity is faster, the action potential overshoot is greater, the maximum diastolic potential is more negative, the action potential is shorter, and the spontaneous rate is faster than in balls from the centre (Fig. 7) [63,67,87,89,90]. Most of these differences (except of spontaneous rate) are concordant with the regional differences in action potential configuration in the intact SA node (Fig. 3). In a recent study, in which the action potential characteristics of small balls of tissue from more superior and more inferior regions as well as the periphery and centre of the rabbit SA node were compared, the periphery–centre differences described above were shown to be just one component of a complex two-dimensional variation in these parameters [65]. In the small balls of tissues (especially from the periphery), there was a decrease in maximum upstroke velocity, an increase of action potential duration and a decrease in spontaneous rate from the more superior tissue to the more inferior tissue. These changes (except of spontaneous rate) are again concordant with regional differences in the intact SA node.
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The one discrepancy between small balls of SA node tissue and the intact SA node concerns pacemaker activity. The intrinsic pacemaker activity of small balls of tissue taken from different regions of the SA node is greater in balls from the periphery than in balls from the centre (Fig. 7). This paradox, which was also shown by Opthof et al. [91] in small pieces of rabbit SA node tissue, can be explained by the electrotonic influence of the atrial muscle. The periphery of the SA node is connected to a large mass of atrial muscle in the crista terminalis through gap junctions. The pacemaker depolarization in peripheral SA node cells will, therefore, be reduced by the hyperpolarizing current flowing from the non-pacemaking atrial cells, which have more negative diastolic potentials. The cells near the centre of the SA node may be subjected to less electrotonic interference from the non-pacemaking atrial muscle because of their greater distance from the crista terminalis and the poorer electrical coupling between cells. Evidence to support this idea was presented by Kirchhof et al. [92]. They showed that, when the atrial muscle was cut away from the SA node, the leading pacemaker site shifted from the centre to the periphery of the SA node and there was an increase in the spontaneous rate of the preparation. In a modelling study using a massively parallel computer, Winslow et al. [93] examined the spontaneous activation pattern in the SA node and surrounding atrial muscle in a multicellular model, in which the regional differences in electrical activity of small balls of SA node tissue [87] were incorporated. When the SA node was not connected to the non-pacemaking atrial cell network, the action potential was initiated in the periphery of the node, but when it was connected to the atrial cell network, the leading pacemaker site was shifted from the periphery to the centre of the SA node and there was a decrease in the spontaneous rate. Similar results were obtained by Boyett et al. [94] using a multicellular model of the SA node and surrounding atrial muscle. In experiments using an external circuit that mimics the gap junction conductance (Gc), Watanabe et al. [95] demonstrated that the spontaneous activity of rabbit SA node cells was easily inhibited when they were connected to a membrane model (resistance–capacitance circuit) of an atrial cell even at relatively low Gc, >580 pS/cell (less than measured Gc — Section 3.2).
In the intact SA node of rabbit, Kerr et al. [96] found that the amplitude of a premature action potential deceased progressively from the periphery to the centre of the SA node. They, therefore, suggested that there is a progressive gradation of refractoriness from the periphery to the centre. Kodama and Boyett [87] applied premature stimuli to small balls of rabbit SA node tissue. The strength–interval curve was shifted upwards and to the right if the ball was more distant from the crista terminalis and closer to the centre of the SA node. The restitution curves of upstroke velocity and amplitude of premature action potentials showed a similar rightward shift, indicating slower recovery of excitability in balls from closer to the centre of the SA node. Such gradation of refractoriness is most likely the consequence of the different ionic currents responsible for excitation in the periphery and the centre of the SA node: iNa and iCa,L, respectively, as described in later sections. iCa,L requires a much longer time for repriming (reactivation) as compared to iNa [97,98].
6.2 Block of iNa and iCa,L
In the intact SA node, there is a gradual decline in the upstroke velocity of the action potential from the periphery to the centre (Section 4). Lipsius and Vassalle [99] and Kreitner [62] observed that in the intact SA node of the guinea-pig and rabbit tetrodotoxin (TTX), a selective iNa blocker, reduced the upstroke velocity in the periphery but not in the centre. In contrast, Verheijck et al. [100], Kodama et al. [90] and Yamamoto et al. [61] showed that, in the intact SA node of the rabbit, nifedipine, a potent iCa,L blocker, inhibited or abolished the action potential in the centre, whereas the action potential in the periphery was well preserved in the presence of the compound.
Kodama et al. [90] investigated the effect of block of iNa by TTX on small balls of rabbit SA node tissue. In the example shown in Fig. 8A, in the peripheral ball (left), the maximum upstroke velocity of the action potential was 100 V/s, and TTX reduced it to 5 V/s. TTX also reduced the take-off potential from –61 to –40 mV and this resulted in a substantial increase in cycle length. In the central ball, the maximum upstroke velocity was 4 V/s and TTX had no discernible effect on electrical activity (Fig. 8A, right). Under control conditions there were decreases in the take-off potential and maximum upstroke velocity in balls from the periphery to the centre (Fig. 7). In the presence of TTX, these gradients were abolished due to a significant reduction of the take-off potential and the maximum upstroke velocity in balls from the periphery [90]. Kodama et al. [90] also investigated the effect of block of iCa,L by nifedipine on small balls of rabbit SA node tissue. Verheijck et al. [101] have shown that nifedipine (5 µM) selectively blocks iCa,L of rabbit SA node cells without affecting iNa, T-type Ca2+ current (iCa,T), delayed rectifier K+ current (iK) and hyperpolarization-activated current (if). In the example shown in Fig. 8B, on application of nifedipine, the action potential of the central ball was abolished (Fig. 8B, right). In marked contrast, nifedipine failed to abolish the action potential in the peripheral ball; instead the cycle length was shortened (Fig. 8B, left). This was the result of a shortening of the action potential and an increase in the pacemaker slope. In the presence of 2 µM nifedipine, on average, the rate was increased in ball A by 21%, increased or decreased in ball B, decreased in balls C and D by 86 and 78%, respectively, and abolished in ball E. Zaza et al. [102] showed in rabbit SA node cells that nifedipine-sensitive current is outward during the pacemaker potential, possibly as a result of a decrease in a Ca2+-activated outward K+ current. This finding is compatible with the observation in Fig. 8B, and may help explain the chronotropic effect of nifedipine.
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These results show regional differences in the role of iNa and iCa,L in pacemaker activity of the SA node and Kodama et al. [90] suggested that iNa is responsible for the action potential upstroke in the periphery, whereas iCa,L is responsible in the centre. This can explain why the take-off potential and upstroke velocity are higher in the periphery and lower in the centre (Fig. 7B) (the threshold of iNa is higher than that of iCa,L), why block of iNa affects pacemaker activity in the periphery but not the centre, and why block of iCa,L abolishes pacemaker activity in the centre and not the periphery. However, the two currents do not play identical roles: in the centre, block of iCa,L leads to the abolition of the action potential, whereas in the periphery, block of iNa does not lead to the loss of the action potential [90]. This is presumably because in the periphery iCa,L takes over the role played by iNa after block of iNa (iCa,L is present in both the centre and periphery).
In the rabbit SA node, Verheijck et al. [29] reported that there exists a substantial number of atrial cells (as well as SA node cells) even in the centre of the SA node, and the ratio of atrial cells:SA node cells gradually increases from the centre of the SA node (ratio, 41:59) to the periphery (ratio, 63:27). Atrial cells in the SA node has also been reported by others [21,57,60]. Verheijck et al. [29] also reported that morphological differences in the SA node cells are not associated with differences in action potential configuration and pacemaker activity (contrary to our own findings — Section 8). Based on these data, Verheijck et al. [29] hypothesized a ‘mosaic model’ (Fig. 9) in which there is a random mix of the two cell types and the ratio of atrial cells:SA node cells increases from the centre to the periphery of the SA node. If iNa is present in atrial cells but not in SA node cells, this will explain the regional variation in the roles of iNa and iCa,L. Our observations on single cells isolated from the rabbit SA node, however, suggest a ‘gradient model’ (Fig. 9), because we do not observe a significant number of atrial cells amongst cells isolated from the SA node and we do find morphological differences in SA node cells are associated with differences in action potential configuration and pacemaker activity (Section 8). According to the gradient model, there is a progressive regional variation in the properties of SA node cells from the centre to the periphery (Fig. 9). According to this model, the regional variation in the roles of iNa and iCa,L can be explained by a decline in the expression of Na+ channels from the periphery to the centre. Evidence for this is presented in Section 8. A greater density of iNa in the periphery may be functionally important: it may protect the SA node from the hyperpolarizing influence of the surrounding atrial muscle, because hyperpolarization will lead to a reduction of inactivation of iNa and this may help overcome the effects of hyperpolarization. iNa in the periphery may also help the SA node to drive the atrial muscle: using a multicellular model of the SA node, Zhang et al. [103] showed that the elimination of iNa from the periphery of the SA node resulted in SA node exit block (i.e. the SA node was no longer able to drive the atrial muscle).
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6.3 Block of 4-AP-sensitive current
It is well known that transient outward current (ito) is present in atrial and ventricular cells and plays an important role in the early repolarization phase of the action potential [104]. ito is known to be blocked by 4-aminopyridine (4-AP). ito is also known to be present in the SA node [30,105–108]. Boyett et al. [63] examined the effects of 5 mM 4-AP on small balls of rabbit SA node tissue. 4-AP increased the duration of the action potential and the increase was greater in peripheral tissue than in central tissue: 42–63% in balls A and B from the periphery and 21–22% in balls D and E from the centre. 4-AP also altered the spontaneous cycle length, increasing it in peripheral tissue (by 13–28% in balls A and B) but decreasing it in central tissue (by 5–26% in balls D and E). There is an increase in action potential duration from the periphery to the centre under normal conditions (Fig. 7) and it has been argued that this is a protective mechanism (Section 4.1) — this increase in action potential duration was no longer significant in the presence of 4-AP [63], which suggests that 4-AP-sensitive current is, in part at least, responsible for it. In peripheral tissue, action potentials with notches are often observed (a brief period of rapid repolarization after the action potential upstroke perhaps followed by a second period of depolarization); the notches were abolished by 4-AP in the study of Boyett et al. [63].
Boyett et al. [63] also examined regional differences in the effects of 4-AP in the superior–inferior direction in four strands of balls of tissue. In transitional/central balls (C and D), the average percentage increase in action potential duration was significantly greater in tissue from a more inferior region: 23% in strand 1 (superior) and 86% in strand 4 (inferior). 4-AP caused no significant change in cycle length in balls from strands 1–3, but significantly increased cycle length (by 30%) in balls from strand 4 from the more inferior region. These results suggest that the 4-AP sensitive currents (itrans and isus: ito and possibly iK,ur — Section 8.2) play a more important role in the periphery of the SA node than the centre, and in the more inferior region of the SA node than the more superior region. There are two possible reasons for the regional differences in the effects of 4-AP. First, the density of 4-AP-sensitive current may be greater in SA node cells in the periphery than in the centre (gradient model). Secondly, because the maximum diastolic potential is more negative in the periphery of the SA node than in the centre, the voltage-dependent inactivation of ito during diastole is expected to be less in the periphery. Voltage-clamp experiments on rabbit SA node cells by Honjo et al. [108] and Lei et al. [106] provide evidence for the former interpretation (Section 8.2).