J. Smooth Muscle Res. (2003) 39 (5): 175–193 175

Mini Review Sino-Atrial Nodal Cells of Mammalian : Ionic Currents and Gene Expression of Pacemaker Ionic Channels

Hiroyasu SATOH1

1Department of Pharmacology, Division of Molecular and Cellular Biology, Nara Medical University, Kashihara, Nara 634-8521, Japan

Abstract The is a sino-atrial (SA) nodal cell. The signal induced by this pacemaker is distributed over the surface by a specialised conduction system and is clinically recorded as the ECG. The SA nodal cells are highly resistant to cardiac failure and ischemia. Under calcium overload conditions, some dysrythmias of SA nodal cells occur easily. Morphological analysis under these conditions shows swelling of the cisternae of the Golgi apparatus, with little or no other histological change or damage being observed. The rate of sinus rhythm is quite different between various species. The investigations of SA nodal cells have so far clarified the pacemaker mechanisms involved. A number of ionic channel currents or pacemaker currents, contribute to the depolarization of the (phase 4). This will not occur with a single current. Recent experiments have identified several novel pacemaker currents and have also revealed several differences in the pacemaker currents between species. The

marked hyperpolarization-activated inward current (If) appears in SA nodal cells of most + species, while the inwardly rectifying K current (IK1) with masked If current is found in

those of the rat and monkey. In addition, the rapidly activated current (IKr) and slowly + activated current (IKs) of the delayed rectifier K current (IK) contribute to the pacemaker

potential in guinea pig SA nodal cells, with only the IKs current in porcine SA nodal cells

and only the IKr current in the rat and rabbit. These differences in ionic channels presumably result from differences in gene expression. Some smooth muscle cells also possess the capacity to beat spontaneously. Uterine smooth muscle cells also exhibit an

If current. The basal mechanism for spontaneous activity in both SA nodal cells and smooth muscle cells is almost the same, but some differences in the ionic channels and their genetic expression may contribute to their respective pacemaker currents.

Key words: Pacemaker currents, Ionic channels, Heart rate, Gene expressions, Sino- atrial nodal cells

Correspondence to: H. Satoh, Department of Pharmacology, Division of Molecular and Cellular Biology, Nara Medical University, Kashihara, Nara 634-8521, Japan Phone: +81-744-29-8831 Fax: +81-744-25-7657 e-mail: [email protected] 176 H. SATOH

Introduction

The sino-atrial (SA) node is known as the pacemaker region of the mammalian heart. In previous experiments using SA nodal cells, we have shown that dysrhythmias (or arrhythmias) are easily elicited under conditions involving calcium overload that occur during ischemia and cardiac failure. Clinically these SA nodal dysfunctions cause bradyarrhythmias in general and are associated with syncope but rarely with death. In SA nodal cells, spontaneous beating or contractions is associated with interactions 2+ + between ionic currents such as the L-type Ca (ICaL), the delayed rectifier K (IK) and the hyperpolarization-activated inward (If) currents (Noble, 1984; Guo et al., 1995; Mitsuiye et al., 2000). Noma and colleagues (Guo et al., 1997; Shinagawa et al., 2000; Mitsuiye et al., 2000) have more recently demonstrated additional underlying currents in rat SA nodal cells, namely the + rapidly activated K current (IKr) and the sustained inward current (Ist). These currents are major pacemaker currents in addition to the ICaL and If currents. Our recent experiments have revealed some differences in the pacemaker currents found in different species. Specific differences in ionic channel distribution and their gene expression may exist, presumably resulting in differences in the sinus rate and drug sensitivity between species.

Localization and Histology

The SA node region is located on the endocardial surface at the edge of the right atrium, bounded on two sides by the superior and inferior venae cavae and around the crista terminalis between the venae cavae and the right atrial muscle. Under a lower power microscope, the SA node appears as a translucent muscular region near the sino-atrial node artery. The most prominent feature is the ring bundle, which is a thin flap of tissue that extends around most of the periphery of the node and that usually appears to be the most vigorously beating part in an isolated node. Under the electron microscope, SA nodal cells are observed to have a relatively large nucleus and a few myofilaments (Satoh and Uchida, 1993). There are many caveolar invaginations along the surface membranes of these cells (Masson-Pevet, 1979). The intercellular space at 20 nm is wide compared with that observed in other tissues. Isolated SA nodal cells are spindle- or spider-shaped and have a maximum length of 25–30 µm, with an irregular profile in cross section and a diameter of less than 8 µm. Isolated spontaneously beating SA nodal myocytes are curved and not flat on their base (Shinagawa et al., 2000).

Differences between Species

Determinants of sinus rate The sinus rate of spontaneously beating SA nodal cells is said to be dependent on body weight (or size) or on the stage of pregnancy (Opthof, 2001). In isolated hearts, the sinus rate is extremely fast in small animals (i.e., mouse, rat and rabbit), whereas it is slower in large Cardiac pacemaker currents 177

Table 1 Electrophysiological parameters of isolated pacemaker cells from different species

CL APA MDP APD50 Vmax (ms) (mV) (mV) (ms) (V/s) mouse 144 63 –60 82 6.2 rat 282 73 –58 88 4.4 guinea pig 295 76 –59 88 3.6 rabbit29876–6511010 cat41860–592022.8 monkey 634 65 –55 186 3.6 pig 784 75 –59 197 2.7 human 2300 72 –62 309 5.4 APA: action potential amplitude. MDP: maximum diastolic

potential. APD50: 50% repolarization of action potential duration. CL: cycle length. animals (i.e., human and elephant) (Table 1). It seems possible that the total number of spontaneous beats over the life span might be related to the metabolic rate of the animal concerned, as well as being dependent on body weight. As a result, small animals would exhibit a faster sinus rate and have a shorter life span, although this is not clear yet. Boyett et al. (2000) have recently reported that the beating rate of cells from the rabbit SA node depends on the cell size. In addition, the phase 4 slope and Vmax are dependent on the cell capacitance (22.0 to 57.5 pF). In general, increasing the cell capacitance enhances the amplitude of the ionic channel currents, with a simultaneous increase in the sinus rate. If the cell size of an animal was related to its body weight, large animals might possess larger SA node cell sizes and thus exhibit higher sinus rates. However, this relationship is quite the opposite as larger animals possess a slower sinus rate. Also, larger animals such as the monkey and the pig do not always possess larger sized SA nodal cells. Actually, we have found no marked difference in cell size between species, although we have not examined the full range of animals in our experiments. But we conclude that there is no relationship between the SA node cell size and the body weight of different species.

Action potentials Spontaneous action potentials in SA nodal cells possess several significant characteristics. When compared with the action potentials of atrial or ventricular cardiomyocytes, the SA nodal cell action potential is characterized by a pacemaker potential, seen as a slow depolarization during phase 4 diastole, which then elicits a spontaneous action potential. This is then repeated successively (Fig. 1A). In addition, a slow depolarization in phase 0 and a lack of phase 1 are observed. The maximum diastolic potential (MDP) was –50 to –60 mV and much lower than + other cardiomyocytes, indicative of the minor contribution of the Na channel current (INa), because the INa is activated at around –80 to –90 mV. To generate spontaneous action potentials, it is necessary for the membrane to depolarize beyond threshold potential during the diastole of phase 4. The rate of spontaneous beating is modulated by (a) the rate of pacemaker depolarization, (b) the MDP, (c) the threshold potential, and (d) the action potential duration (APD). These factors are mostly regulated by pacemaker 178 H. SATOH

Fig. 1. Action potentials and ionic currents of spontaneously beating sino-atrial (SA) nodal cells of the rat. A, Spontaneous action potentials. B, Ionic currents resulting from depolarizing pulses between –20 to 60 mV, and also from hyperpolarizing pulses between –40 to –120 mV. HP was –30 mV. currents (which form the pacemaker potential) which flow during diastole, which is contributed to by a number of ionic channel currents, as discussed below.

Pacemaker currents A number of different time-dependent currents contribute to the pacemaker activity of the SA nodal cells (Fig. 1B). In general, the regulation of the spontaneous beating SA nodal cells is considered to be due to (a) the ICaL current, (b) the time-dependent decay (deactivation) of IK conductance, and (c) the If current (Noble, 1984; Irisawa et al., 1993).

The ICaL is the most important , because the generation of the pacemaker potential of SA nodal cells is almost at the same potential as the threshold potential for ICaL channels. Ca2+ antagonists easily stop the spontaneous action potentials. At lower concentrations these antagonists suppressed and slowed just the last part of the pacemaker depolarization (Satoh and Tsuchida, 1993). A similar phenomenon was observed soon after the application of these antagonists. 2+ The contribution of the T-type Ca current (ICaT) to pacemaking is small, and is limited to approximately the first third of the pacemaker potential (Noble, 1984), because the ICaT current starts to activate at around –80 mV, which is similar to the INa channel. This is also supported by Cardiac pacemaker currents 179

our experiments using ICaT channel inhibitors (Satoh, 1995a). + The delayed rectifier K channel (IK) is responsible for repolarization, and adjusts the resting potential (RP) after each action potential. The IK is comprised of three separate channels; the slowly activated (IKs), rapidly activated (IKr), and ultrarapidly activated (IKur) channel currents. The IK current is markedly activated during the repolarization phase (phase 3) of the action potentials, and as a result, the myocyte repolarizes back down to the RP. After repolarization during diastole, the IK slowly deactivates, which is most important for the pacemaker potential. The deactivation leads to a gradual increase in the permeability ratio of

PNa/PK of the membrane. This increase potentiates the pacemaker depolarization. More recently, the pacemaker mechanism of SA nodal cells has also been reported to involve IKr (Guo et al., 1995; Shinagawa et al., 2000; Matsuura et al., 2002). When IKr is blocked by E-4031, the spontaneous activity is depressed and sinus arrest often occurs in both rat and guinea pig SA nodal cells.

The sustained inward current (Ist) is also specific to SA nodal cells and is closely related to

ICaL (Mitsuiye et al., 2000). The Ist is the key pacemaker current which generates the diastolic depolarization (Guo et al., 1997; Shinagawa et al., 2000). The current is nicardipine-sensitive with a conductance of 13 pS Na+ current. The current density is very small (2 pA/pF) in rabbit 2+ SA nodal cells (Mitsuiye 2000). The Ist is resistant to TTX (30 µM) but is highly sensitive to Ca antagonists such as by nicardipine (0.25 to 0.5 µM) and verapamil (1 µM). Bay K 8644, a Ca2+ channel opener, enhances the Ist. Thus, Ist activation makes a major contribution to the pacemaker depolarization.

The If current is “specific” to pacemaker nodal cells and is generally found in cells with spontaneous activity. It seems unlikely that this current largely contributes to pacemaking activity under normal conditions, because full activation in voltage-clamp experiments needs a greater hyperpolarization (over –70 mV) and longer duration (over 1 s) of the stimulation pulse. In the SA nodal action potentials, the MDP is approximately –60 to –70 mV and the 50% repolarization of APD (APD50) is around 80 to 300 ms. Therefore, inhibition of the If current (i.e., by Cs+) would exhibit only a minor effect on the spontaneous activity of SA nodal cells. The rate of pacemaker activity is strongly regulated by the autonomic nervous system. β- Adrenoceptor stimulation causes a marked positive chronotropic effect due to enhancement of + ICaL, If, Ist, and IKs ionic channels. On the other hand, the opening of muscarinic K channels stimulates a substantial outward current. The hyperpolarizing shift of the MDP induced by IKACh activation causes a negative chronotropic effect. Simultaneously, ACh reduces ICaL and If. Thus, the pacemaker potential is regulated by a complicated interaction of multiple currents under the regulation of the autonomic nervous system and is not due to a single current. Even if a particular current makes a reduced or no contribution under normal conditions, it might play a role in the generation of pacemaker depolarization and the maintenance of regular rhythm under abnormal (disease) conditions.

Role of the Hyperpolarization-activated Inward Current (If)

One of the most striking features of SA nodal cells is the If (funny) current. The inward 180 H. SATOH

Fig. 2. Pacemaker ionic currents recorded from SA nodal cells of different species. Holding potential was –40 mV. The duration of test pulses was 1 s. A, rabbit; B, guinea pig; C, rat. Note that after application of 2+ Ba (2 mM), an If current was unmasked.

flowing If current is markedly activated by a hyperpolarizing pulse. There is considerable debate over the exact role of this current. The If may increase the speed of onset of pacemaker depolarization. As mentioned above, the If current makes only a minor contribution to the pacemaker depolarization. However, cells that do not beat spontaneously do not exhibit the If.

Therefore, we now consider that the If current may contribute somewhat indirectly to the diastolic depolarization involved in spontaneous beating. 2+ The If activation depends critically on the levels of cytosolic Ca (Hagiwara et al., 1988; 2+ Satoh, 2003). In normal cells with a cytosolic Ca of 100 nM, the activation curve for If lies between –40 and –80 mV, suggesting that in SA node cells the If could contribute to a substantial steady inward current within the pacemaker range (–65 to –45 mV). Cs+ ions are relatively + selective blockers of the If current. In the presence of 2 mM Cs which fully blocks the If, SA nodal cells do not cease beating spontaneously (Denyer and Brown, 1990; unpublished data).

This result emphasizes that the If current is not essential for pacemaking.

In rabbit and guinea pig SA nodal cells, hyperpolarizing pulses activate marked If currents

(Fig. 2A-B), whereas in rat SA nodal cells they do not always activate an If current (Fig. 2C).

However, even cells without an If current can still beat spontaneously (Shinagawa et al., 2000).

In mouse SA nodal cells, the functional properties of the If current resemble those of the If current in rabbit SA nodal cells (Matteo et al., 2001). In monkey SA nodal cells, as in rat SA nodal cells, the If current is not common even in spontaneously beating cells or typically beating spider-shaped cells (unpublished data). Therefore, the role of the If current seems likely to be Cardiac pacemaker currents 181

Fig. 3. Existence of IKr and IKs in rat SA nodal cells. A–B, Control and in the presence of E- 4031. C, Activation curves for IKr. D, Activation curves for IKs.

that of rate modulation rather than of the generation of pacemaker depolarization. But SA nodal cells that are not spontaneously beating lack an If current. In young (3–5 day old) embryonic chick ventricular cardiomyocytes, strong spontaneous beats appear, and simultaneously a marked If current is also identified (Satoh and Sperelakis, 1991), but both of these decrease and disappear with further embryonic development.

Dependence on IKr or IKs

+ The delayed K current in SA nodal cells is comprised of both IKr and IKs (Sanguinetti and

Jurkiewicz, 1990). The IKr is rapidly activated within 200 ms while IKs is slowly activated in 1 to 2 s. As shown in Fig. 3, both currents are present in rat SA nodal cells (Satoh, 1999; Shinagawa et al., 2000; Ono et al., 2000; Matsuura et al., 2002). The IKr inhibition may cause a more positive

MDP and a longer APD. Thus, it is likely that the sinus rate may be regulated by IKr and/or IKs. The time-dependence of activation would be dependent on the sinus rate. The fast sinus rate found in small animals might be driven by IKr, whereas the slow sinus rate found in larger animals might be driven by IKs. The IKs begins weakly at first during repolarization and then 182 H. SATOH

Fig. 4. Modulation by E-4031 of the spontaneous action potentials in rat SA nodal cells. A,

Traces of the action potentials, the Vmax and the sinus rate. B, Faster traces of the action potentials and the Vmax as recorded at the points (a–c) indicated in panel A.

gradually builds up. The rectification is delayed until the rapid phase of repolarization. The IKs channel is sensitive to β-adrenergic stimulation. On the other hand, the IKr rapidly activates to maximum intensity, then levels off. The IKr channel is insensitive to β-adrenergic stimulation, but is blocked by many antiarrhythmic drugs (e.g. disopyramide and flecainide). The IKs blockade is limited to several arrhythmic drugs such as cibenzoline and amiodarone. As shown in Fig. 4, the spontaneous action potentials in rat SA nodal cells are suppressed by

3–5 µM E-4031 which blocks IKr. On the other hand, 293B (30 µM) which blocks IKs has a much smaller or no effect (Fig. 5). Both of these responses are observed in rat and rabbit SA nodal cells (Shinagawa et al., 2000; Lei et al., 2002) (Table 2). Therefore, the contribution of IKs to spontaneous action potentials is small under normal conditions. On the other hand, in guinea pig SA nodal cells, both IKr and IKs currents play crucial roles in the spontaneous electrical activity of these cells (Matsuura et al., 2002). In contrast, the spontaneous activity in porcine SA nodal cells depends on IKs alone and not on IKr (Ono et al., 2000). This discrepancy between different species is inexplicable at this time. Anyway, it appears from these results that spontaneous action potentials are dependent on IKr or/and IKs, but that the sinus rate is necessarily independent of these currents.

Other Pacemaker Currents

In general, there are minor pacemaker currents that contribute somewhat to the pacemaker Cardiac pacemaker currents 183

Fig. 5. Modulation by 293B of the spontaneous action potentials in rat SA nodal cells. A,

Traces of the action potentials, the Vmax and the sinus rate. B, Faster traces of the action potentials and the Vmax as recorded at the points (a–d) indicated in panel A.

Table 2 Dependence of IKr and IKs for Table 3 Dependence on If and IK1 for spontaneous activity in SA spontaneous activity in different nodal cells of different species species

IKr IKs If IK1 mouse + – mouse +/– + rat + – rat +/– + guinea pig + + guinea pig + – rabbit + – rabbit + – pig – + pig + – monkey ? ? monkey +/– + depolarization. These currents may contribute more significantly to pacemaker depolarization under some disease conditions. + The inward rectifying K (IK1) channel is voltage-dependent and maintains the RP. It is well known that IK1 is usually absent in the SA nodal cells of rabbit and guinea pig (Fig. 2A-B), but it is present in both atrial and ventricular myocytes of these species. Actually, the IK1 is not present in the SA nodal cells of most species (Table 3). The lack of IK1 may make the membrane potential unstable after repolarization and exert a positive MDP in SA nodal cells. In rat and monkey SA nodal cells, however, a marked IK1 actually appeared (Fig. 2C). After application of 2+ Mg (0.5 to 2 mM) to block the IK1, a masked If current was revealed (Shinagawa et al., 2000).

These results demonstrate strongly that the IK1 makes little or no contribution to pacemaker depolarization.

The transient outward current (Ito) plays an important role in action potential repolarization 184 H. SATOH

Table 4 Gene expression of the pacemaker ionic channels in sino-atrial nodal cells α Subunits β Subunits Current Protein Gene chromosome Gene chromosome Major channels

ICaL Cav1.2 α1C(CACNA1C) 12pter13.3 β1(CACNB1) 17q21-22 β2(CACNB2) 10p12 α2δ(CACNA2D1) 7q21-22 IKr ERG KCNH2 7q35–36 MiRP1 KCNE2 21q22.12 IKs KvLQT1 KCNQ1 11p15.5 minK KCNE1 21q22 If HCN1 BCNG-1 HCN4 HCN4 15q24–25

ICaT Cav3.2 α1H(CACNA1H) 16p13.3 Cav3.1 α1G(CACNA1G) 17q22 Minor channels

IK1 Kir2.1 KCNJ2 17q Kir2.2 KCNJ12 17p11.2

IKACh Kir3.1 KCNJ3 2q24.1 Kir3.4 KCNJ5 11q24

IKATP Kir6.2 KCNJ11 11p15.1 SUR2A ABCC9 12p12.1 KCNJ5 11q24–25

Ito Kv4.3 KCND3 1p13.3 KChIP2 10q24 Kv1.4 KCNA4 11q14

INa Nav1.5 H1(SCN5A) 3p21-24 β1(SCN1B) 19q13.1–13.2 β2(SCN2B) 11q23 IClCa CIC4 CLCN4 RyR RyR2 1q42–43

IP3RIP3R1 INa/Ca NCX1 NCX1.1–1.2 Ibg TW1K1 KCNK1 15q24–25 CFTR ABCC7 7q31.2 KvLQT1 KCNQ1 11p15.5 MiRP1 KCNE2 21q22.12

IGJ Cx45, Cx40

and contributes to cardiac electrical heterogeneity (Campbell et al., 1995). The expression of the Ito current is heterogenous (Lei et al., 2000; Decher et al., 2001; Rosati et al., 2001). The Ito is characterized by rapid activation and inactivation. The Ito has 6 segments per domain, + composed of two (Ito1 and Ito2) channels. Ito1 is a voltage-dependent K channel and initiates repolarization. A smaller sustained Ito component may also contribute to the APD 2+ (repolarization) in SA nodal cells. Ito2 is a chloride current which is activated by Ca (and is thus often termed IClCa).

IKACh is one of the ligand-activated channels directly activated by the acetylcholine (ACh) released from parasympathetic nerves. ACh also modulates these ionic channels due to mediation through muscarinic (M2) receptors on the cardiac cell membranes. The SA node is precisely controlled by the autonomic nervous system with the regular rhythm largely regulated through stimulation of M2 receptors. ACh activates IKACh channels during rapid repolarization and during pacemaker depolarization. Its activation lowers the MDP to a more negative potential. The gene expression of IKACh is encoded by a complex containing Kir3.1 and Kir3.4 subunits (Krapivinsky et al., 1995) (Table 4). Kir3.1 and M2 receptor proteins are colocalized.

IKATP channels can open under conditions of reduced cellular ATP levels (1 mM) such as Cardiac pacemaker currents 185 occurs in cardiac failure and ischemia. Thus, this is a cardioprotective channel. The openers of these channels (cromakalim, pinacidil and nicorandil) caused a negative chronotropic effect that prolonged the APD in rabbit SA nodal cells (Satoh and Hashimoto, 1984; Satoh, 1993). These drugs inhibit ICa but fail to affect IK. In our laboratory, inhibition of ATP production with cyanide

(5 mM), which may activate IKATP channels, causes a negative chronotropic effect but without the occurrence of sinus arrest. Thus, IKATP channels would also contribute somewhat to pacemaker depolarization.

INa initiates the phase 0 depolarization of cardiomyocytes and of cardiac .

However, application of TTX, a selective blocker of INa channel, fails to alter the generation of spontaneous action potentials. The INa may contribute to subsidiary pacemaker activity in peripheral regions, providing a backup mechanism (Warth et al., 1996; Kodama et al., 1997;

Wang et al., 1998). The INa has not been found consistently in SA nodal pacemaker cells. The behavior of INa is somewhat similar to that of ICaT and thus contributes little to diastolic depolarization.

The background (or non-specific) inward current (Ibg) produces a gradual depolarization of the membrane during the pacemaker potential (Wilders et al., 1991; Demir et al., 1994). Sodium and (ICl) currents are mostly known (Seyama, 1976; Hagiwara et al., 1992). The Na-K pump current and the Na-Ca exchanger current may play roles in the maintenance 2+ – and modulation of pacemaking. The Ca -activated Cl current (IClCa), while not always present in rabbit SA nodal cells (only found in approximately one third of the cells), is activated during the pacemaker cycle (Verkerk et al., 2002). The reversal potential for Cl– ions of cardiomyocytes is approximately –50 mV (Sorota, 1999). Thus, IClCa activation generates an inward depolarizing current during the pacemaker potential and generates an outward repolarizing current during phases 2 and 3 of the SA nodal action potentials.

Cytosolic Ca2+

The cyclical changes in electrophysiological activity, which produce the spontaneous beats, originate as a result of the opening and closing of time- and voltage-dependent ionic channels in the sarcolemmal membrane. In addition, the establishment of this rhythm may be considered to be triggered and controlled by oscillations in the levels of cytosolic Ca2+ due to the release of Ca2+ from the SR (Tunwell et al., 1996). A mechanism has been found that involves T-type Ca2+ channels triggering transient Ca2+ release or Ca2+ sparks (Fabiato, 1992). These Ca2+ sparks play a prominent role in bringing the pacemaker potential to threshold in spontaneously beating atrial cells of the cat (Zhou and Lipsius, 1993; Lipsius et al., 2001). Ca2+ release during the pacemaker potential may possibly depolarize the pacemaker potential by stimulating the inward Na+/Ca2+ exchange current. In our laboratory, however, switching from perforated patch- clamping to whole-cell patch-clamping (with low EGTA concentrations in the pipette solution) largely failed to affect spontaneous action potentials. Even when cytosolic Ca2+ is not fully chelated at low concentrations of cellular EGTA (20 µM), spontaneous beats are suppressed but never abolished and the APD become relatively longer (unpublished data). In spontaneously beating embryonic chick cardiomyocytes, the sinus rate increases with an 186 H. SATOH increase in pCa levels (in a range from pCa 10 to pCa 6) (Satoh, 1995b). In rabbit SA nodal cells, however, ryanodine and thapsigargin have little or no effect on chronotropism. In guinea pig ventricular muscle cells, thapsigargin had a negative inotropic effect (Nario and Satoh, 1996) while in rabbit SA nodal cells, ryanodine at high concentrations caused a negative chronotropic effect (Satoh, 1997). Therefore, we conclude that in SA nodal cells, cytosolic Ca2+ (Ca2+ sparks) makes only a minor contribution to pacemaker depolarization.

Molecular and Gene Expression

The gene expression of the main pacemaker channels is described below, however not all ionic channels are discussed in this section.

T- and L-type Ca2+ channels For the low voltage-activated Ca2+ channels (Cav), Cav3.1 and Cav3.2 encode α subunits of

ICaT (Cribbs et al., 1998). The gene expression of channels is summarized in Table 4. The most 2+ significant expression of the Ca channel is Cav3.1 (α1G). Cav3.1 mRNA expression is 30-fold greater in the SA nodal cells of the mouse than in the atrial cells (Bohn et al., 2000). Cav3.2

(α1H) is present at a moderate level (Bohn et al., 2000). The expression of Cav3.2 is generally lower than that of Cav3.1, but it is relatively higher in SA nodal cells. 2+ On the other hand, the predominant high-voltage activated Ca channel, Cav1.2 (α1C), encodes ICaL. It is more strongly expressed in mouse SA nodal cells than in cells of the atrium (Bohn et al., 2000). The β and α2δ subunits modulate the density, kinetics and activation/ inactivation of the ICaL channel (Catterall, 2000). Also, a small amount of Cav1.3 (α1D) mRNA is detected in mouse SA nodal myocytes. In Cav1.3-knockout mice, however, profound dysrhythmias occur (Platzer et al., 2000). Recently, a role has been established for Cav1.3 Ca2+ channels in the generation of spontaneous activity (Zhang et al., 2002). The half-activation voltage (V1/2) is –17.5 ± 0.9 mV for Cav1.3, and –3.9 ± 1.3 mV for Cav1.2 (Koschak et al., 2001).

If channel

Despite the important physiological functions of If channels, the genes encoding these channels have only recently been identified through heterologous expression (Gauss et al., 1998; Ludwig et al., 1998; Santro et al., 1998). At least four different genes are now recognised. Hyperpolarization-activated cation channels (HCN) contain the families of voltage-gated channels and cyclic nucleotide-gated channels (Kaupp and Seifert, 2001). These channels probably embody six transmembrane segments (S1–S6), with a pore loop between segments S5 and S6 and a cyclic nucleotide-binding domain in the C-terminal region of the polypeptide. Molecular studies have revealed that gene expression encodes HCN1 to 4 (Shi et al., 1999; Ishii et al., 1999; Moroni et al., 2000; 2001; Moosmang et al., 2001). The HCN transcripts are mostly present in SA nodal cells, and are 25 times more abundant in those cells than in Purkinje fiber cells (Shi et al., 1999). The four different mammalian genes involved display high homology. HCN1 is abundantly expressed in the brain, but also in the SA node. HCN2 expression is detected in both the brain and the heart, but is at very low levels in the SA node. HCN3 mRNA Cardiac pacemaker currents 187 is detected in the brain, but at lower levels than the other three subtypes (HCN1, 2 and 4). HCN4 mRNA is detected at high levels in both the brain and the heart. Strong HCN4 expression has also been detected in the rabbit SA node. In addition, moderate amounts of HCN4 mRNA have also been detected in both rat neonatal and adult ventricles and also in human tissue (Ludwig et al., 1999; Shi et al., 1999; Ishii et al., 1999: Moroni et al., 2001).

IK channels

There are two types of IK channels, namely the IKr and IKs channels. The IKr channel is composed of two proteins, the α subunit being ERG and the β subunit being MiRP1. The ERG protein transcript is closely correlated with the presence of IKr in both ferret and rabbit SA nodal cells (Brahmajothi et al., 1997; Wymore et al., 1997). The IKs channel is composed of two different proteins, the α subunit being KvLQT1 and the β subunit being minK. MinK transcripts are more abundant in the SA node than in either the atrium or ventricle (Brahmajothi et al., 1996). It has been reported that a minK-related protein, MiRP1, enhances the density and activation rate of If channels (Yu et al., 2001). MiRP1 mRNA is highly expressed in rabbit SA nodal cells, which suggests that it makes a possible contribution to the pacemaking functions of

SA nodal cells. The details of the IK channels are summarized in Table 4.

The IK1 channel has only two transmembrane segments in each domain. Kir2.1 is expressed as the predominant cardiac IK1 subunit (Brahmajothi et al., 1996). The IKACh channel is formed by complexes containing Kir3.1 and Kir3.4 subunits (Krapivinsky et al., 1995). The protein Kir3.1 is present in SA nodal cells of rat, ferret and guinea pig, while the protein Kir3.4 is found in rat SA nodal cells (Dobrzynski et al., 2001). Cardiac channel expression of IKATP channels is encoded as Kir6.2 and SUR2A (Tucker et al., 1995; Chutkow et al., 1996; Babenko et al., 1998).

IGJ channel Gap-junctional hemichannel (Cx) proteins are the basis of intercellular electrical coupling (Beyer et al., 1990). The SA nodal cells are shielded against hyperpolarizing atrial influences by compartmentalization of Cx expression (Coppen et al., 1999). In the SA nodal cells of both rabbit and human hearts, Cx45 and Cx40 are both expressed, but Cx43 is absent (Ninomiya, 1966; Beyer et al., 1990). Cx40 alone is expressed in 55% of canine SA nodal cells. Cells expressing all 3 types of connexin are located in bundles abutting atrial tissue, whereas Cx40 expressing cells are located in central SA nodal cells (Kwong et al., 1998). Thus, myocytes coexpressing Cx40, Cx43 and Cx45 are found from the SA nodal cells into the atrium, and would transmit pacemaker impulses to drive the atrium.

Myocardial Contraction

Spontaneous beats of the heart are produced as a result of repeated myocardial contractions of SA nodal cells, due to generation of spontaneous action potentials regulated by the underlying ionic channels. Sympathetic stimulation of Ca2+ channels increases the force of myocardial contraction. Simultaneously, the spontaneous beat is stimulated by the enhanced Ca2+ channels (CICR) involved with the SR (both in the exterior cell membrane and in the T tubules). 188 H. SATOH

Fig. 6. Diagrammatic representation of the pendulum movement involved in the repetitive depolarization and a repolarization of the SA nodal cells. The pacemaker potential occurs during the depolarization and plays an important role in the depolarization of the membrane to threshold potential. Visual spontaneous beats are produced by repeated contractions.

Sympathetic stimulation increases the number of Ca2+ ions (mostly of CICR) available for binding to actin TnC sites, resulting in enhancement of the force of myocardial contraction. Once Ca2+ ions are released by actin-myosin dissociation, the excess Ca2+ ions must be quickly removed from the cytosol. Sympathetic stimulation of dormant SRCa2+ ATPase pumps (by G protein phosphorylation) effectively accelerates the transport of Ca2+ from the cytosol into the SR. Phospholamban, a membrane peptide adjacent to some SR Ca2+ ATPase pumps, acts as a brake to inactivate them. The phosphorylation of phospholamban brings about its inactivation of the SR pump; in effect, sympathetic stimulation activates these dormant SR Ca2+ ATPase pumps. Thus, as a result, the excess Ca2+ is removed from the cytosol.

Future Directions, and the Automaticity of Smooth Muscle Cells

The pacemaker mechanisms of SA nodal cells from mammalian hearts can now be understood. Pacemaker activity of the SA nodal cells is a pendulum movement between depolarization and repolarization. The pacemaker depolarization is the initial stages of depolarization followed by phase 0 of the depolarization. These events are summarized in Figure 6. Visual spontaneous beats are the result of repeated contractions. While we are in the process of investigating and elucidating the mechanisms involved in monkey SA nodal cells, it is still unclear whether the same mechanisms are involved in human SA nodal. Further extensive studies are needed to elucidate in more detail the as yet unknown mechanisms involved in spontaneous activity of SA nodal cells (including the molecular and genetic expression of pacemaker ionic channels). It is expected that such a study will be effective and beneficial for Cardiac pacemaker currents 189 the clinical treatment of pacemaker dysfunctions such as sick sinus syndrome. Finally, in smooth muscle cells, the foundation of the pacemaker mechanism is very similar to that found in SA nodal cells. It is again a pendulum movement with a repetitive depolarization and repolarization. In spontaneously beating rat pregnant uterus smooth muscle cells, an If current is identified although not markedly (Satoh, 1995c). It has also been shown that the spontaneous activity of smooth muscle cells may be largely dependent on transient Ca2+ sparks similar to atrial pacemaker cells. The Ca2+ sparks are able to activate Ca2+-dependent K+ channels (IKCa) to produce the repolarization between spontaneous action potentials (Nelson et 2+ al., 1995) as well as Ca -activated Cl- channels (IClCa) to produce the depolarization during pacemaker potential (ZhuGe et al., 1998). The IKCa current in aortic smooth muscle cells is inhibited by β-adrenoceptor and muscarinic receptor stimulation and also by PK-C stimulation (Satoh, 1996). Interstitial cells of Cajal, the gastrointestinal pacemakers, exhibit Ca2+ release 2+ 2+ from IP3-dependent Ca stores (IP3R) activating a Ca -dependent cationic current that drives pacemaker depolarization (Thomsen et al., 1998). The Ca2+ release is sensitive to a variety of second messenger signaling mechanisms in the cytosol. These mechanisms are a potential site for the regulation of spontaneous activity by autonomic neurotransmitters, hormones or drugs.

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(Received August 26, 2003; Acepted September 5, 2003)