Heterogeneous expression of Ca(2+) handling proteins in rabbit sinoatrial node.

We investigated the densities of the L-type Ca(2+) current, i(Ca,L), and various Ca(2+) handling proteins in rabbit sinoatrial (SA) node. The density of i(Ca,L), recorded with the whole-cell patch-clamp technique, varied widely in sinoatrial node cells. The density of i(Ca,L) was significantly (p<0.001) correlated with cell capacitance (measure of cell size) and the density was greater in larger cells (likely to be from the periphery of the SA node) than in smaller cells (likely to be from the center of the SA node). Immunocytochemical labeling of the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger, sarcoplasmic reticulum Ca(2+) release channel (RYR2), and sarcoplasmic reticulum Ca(2+) pump (SERCA2) also varied widely in SA node cells. In all cases there was significantly (p<0.05) denser labeling of cells from the periphery of the SA node than of cells from the center. In contrast, immunocytochemical labeling of the Na(+)-K(+) pump was similar in peripheral and central cells. We conclude that Ca(2+) handling proteins are sparse and poorly organized in the center of the SA node (normally the leading pacemaker site), whereas they are more abundant in the periphery (at the border of the SA node with the surrounding atrial muscle).     

Simulation analysis of excitation conduction in the heart: Propagation of excitation in different tissues

The normal excitation and conduction in the heart are maintained by the coordination between the dynamics of ionic conductance of each cell and the electrical coupling between cells. To examine functional roles of these two factors, we proposed a spatially-discrete model of conduction of excitation in which the individual cells were assumed isopotential. This approximation was reasoned by comparing the apparent space constant with the measured junctional resistance between myocardial cells. We used the four reconstruction models previously reported for five kinds of myocardial cells. Coupling coefficients between adjacent cells were determined quantitatively from the apparent space constants. We first investigated to what extent the pacemaker activity of the sinoatrial node depends on the number and the coupling coefficient of its cells, by using a one-dimensional model system composed of the sinoatrial node cells and the atrial cells. Extensive computer simulation revealed the following two conditions for the pacemaker activity of the sinoatrial node. The number of the sinoatrial node cells and their coupling coefficients must be large enough to provide the atrium with the sufficient electric current flow. The number of the sinoatrial node cells must be large so that the period of the compound system is close to the intrinsic period of the sinoatrial node cell. In this simulation the same sinoatrial node cells produced action potentials of different shapes depending on where they were located in the sinoatrial node. Therefore it seems premature to classify the myocardial cells only from their waveforms obtained by electrical recordings in the compound tissue. Second, we investigated the very slow conduction in the atrioventricular node compared to, for example, the ventricle. This was assumed to be due to the inherent property of the membrane dynamics of the atrioventricular node cell, or to the small value of the coupling coefficient (weak intercellular coupling), or to the electrical load imposed on the atrioventricular node by the Purkinje fibers, because the relatively small atrioventricular node must provide the Purkinje fibers with sufficient electric current flow. Relative contributions of these three factors to the slow conduction were evaluated using the model system composed of only the atrioventricular cells or that composed of the atrioventricular and Purkinje cells. We found that the weak coupling has the strongest effect. In the model system composed of the atrioventricular cells, the propagation failure was not observed even for very small values of the coupling coefficient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/ΔKPQ hearts suggest an overlap syndrome

Mutations in SCN5A, the gene encoding the pore-forming subunit of cardiac Na(+) channels, cause a spectrum of arrhythmic syndromes. Of these, sinoatrial node (SAN) dysfunction occurs in patients with both loss- and gain-of-function SCN5A mutations. We explored for corresponding alterations in SAN function and intracardiac conduction and clarified possible mechanisms underlying these in an established mouse long QT syndrome type 3 model carrying a mutation equivalent to human SCN5A-ΔKPQ. Electrophysiological characterizations of SAN function in living animals and in vitro sinoatrial preparations were compared with cellular SAN and two-dimensional tissue models exploring the consequences of Scn5a+/ΔKPQ mutations. Scn5a+/ΔKPQ mice showed prolonged electrocardiographic QT and corrected QT intervals confirming long QT phenotypes. They showed frequent episodes of sinus bradycardia, sinus pause/arrest, and significantly longer sinus node recovery times, suggesting compromised pacemaker activity compared with wild-type mice. Electrocardiographic waveforms suggested depressed intra-atrial, atrioventricular node, and intraventricular conduction in Scn5a+/ΔKPQ mice. Isolated Scn5a+/ΔKPQ sinoatrial preparations similarly showed lower mean intrinsic heart rates and overall slower conduction through the SAN to the surrounding atrium than did wild-type preparations. Computer simulations of both single SAN cells as well as two-dimensional SAN-atrial models could reproduce the experimental observations of impaired pacemaker and sinoatrial conduction in terms of changes produced by both augmented tail and reduced total Na(+) currents, respectively. In conclusion, the gain-of-function long QT syndrome type 3 murine Scn5a+/ΔKPQ cardiac system, in overlap with corresponding features reported in loss-of-function Na(+) channel mutations, shows compromised SAN pacemaker and conduction function explicable in modeling studies through a combination of augmented tail and reduced peak Na(+) currents.     

用生物学方法重建心脏起搏点的研究进展

正常人的心脏节律源于右心房的天然起搏点（pacemaker）——窦房结。窦房结的功能异常或者房室传导阻滞会导致心率异常（如心律缓慢）。治疗严重的心动过缓需要植入在技术上已经相当成熟的电子起搏器,但这种治疗存在一些缺陷和不足。近年来,在动物实验模型中应用基因或细胞来重建心脏的生物起搏点已经取得了进展。超极化活化环核苷酸门控（hyperpolarization-activated cyclic-nucleotide-modulated,HCN）通道（起搏通道）通过超极化活化的阳离子电流（hyperpolarization-activated cation current,It）调制心脏的自律性。利用病毒载体或转染HCN基因的细胞将HCN基因导入动物心脏内可重建生物起搏点。也有导入其它基因或植入自律细胞来探索心脏起搏点的重建。本文总结了重建心脏生物起搏点的一些研究进展。一旦稳定性和寿命等关键问题得到相应解决,遗传工程改造的生物起搏点可用于治疗严重的心动过缓。     

Conduction barriers and pathways of the sinoatrial pacemaker complex: their role in normal rhythm and atrial arrhythmias

Since Keith and Flack's anatomical discovery of the sinoatrial node (SAN), the primary pacemaker of the heart, the question of how such a small SAN structure can pace the entire heart has remained for a large part unanswered. Recent advances in optical mapping technology have made it possible to unambiguously resolve the origin of excitation and conduction within the animal and human SAN. The combination of high-resolution optical mapping and histological structural analysis reveals that the canine and human SANs are functionally insulated from the surrounding atrial myocardium, except for several critical conduction pathways. Indeed, the SAN as a leading pacemaker requires anatomical (fibrosis, fat, and blood vessels) and/or functional barriers (paucity of connexins) to protect it from the hyperpolarizing influence of the surrounding atrium. The presence of conduction barriers and pathways may help explain how a small cluster of pacemaker cells in the SAN pacemaker complex manages to depolarize different, widely distributed areas of the right atria as evidenced functionally by exit points and breakthroughs. The autonomic nervous system and humoral factors can further regulate conduction through these pathways, affecting pacemaker automaticity and ultimately heart rate. Moreover, the conduction barriers and multiple pathways can form substrates for reentrant activity and thus lead to atrial flutter and fibrillation. This review aims to provide new insight into the function of the SAN pacemaker complex and the interaction between the atrial pacemakers and the surrounding atrial myocardium not only in animal models but also human hearts.     

Reciprocal role of the inward currents ib, Na and i(f) in controlling and stabilizing pacemaker frequency of rabbit sino-atrial node cells.

Experiments and computations were done to clarify the role of the various inward currents in generating and modulating pacemaker frequency. Ionic currents in rabbit single isolated sino-atrial (SA) node cells were measured using the nystatin-permeabilized patch-clamp technique. The results were used to refine the Noble-DiFrancesco-Denyer model of spontaneous pacemaker activity of the SA node. This model was then used to show that the pacemaker frequency is relatively insensitive to the magnitude of the sodium-dependent inward background current ib, Na. This is because reducing ib, Na hyperpolarizes the cell and so activates more hyperpolarizing-activated current, i(f), whereas the converse occurs when ib, Na is increased. The result is that i(f) and ib, Na replace one another and so stabilize nodal pacemaker frequency.     

Importance of Gradients in Membrane Properties and Electrical Coupling in Sinoatrial Node Pacing

The sinoatrial node (SAN) is heterogeneous in terms of cell size, ion channels, current densities, connexins and electrical coupling. For example, Na_v1.5 (responsible for I _Na) and Cx43 (responsible for electrical coupling) are absent from the centre of the SAN (normally the leading pacemaker site), but present in the periphery (at SAN-atrial muscle junction). To test whether the heterogeneity is important for the functioning of the SAN, one- and two-dimensional models of the SAN and surrounding atrial muscle were created. Normal functioning of the SAN (in terms of cycle length, position of leading pacemaker site, conduction times, activation and repolarization sequences and space constants) was observed when, from the centre to the periphery, (i) cell characteristics (cell size and ionic current densities) were changed in a gradient fashion from a central-type (lacking I _Na) to a peripheral-type (possessing I _Na) and (ii) coupling conductance was increased in a gradient fashion. We conclude that the heterogeneous nature of the node is important for its normal functioning. The presence of Na_v1.5 and Cx43 in the periphery may be essential for the node to be able to drive the atrial muscle: Na_v1.5 provides the necessary depolarizing current and Cx43 delivers it to the atrial muscle.     

Simulation of atrial activity by a phase response curve based model of a two-dimensional pacemaker cells array: the transition from a normal activation pattern to atrial fibrillation

In this paper, we present an original model of the atria, based on our hypothesis that atrial cells have features of pacemaker cells, characterized by their normally longer intrinsic cycle lengths and different type of connection (stronger) than the, sino-atrial (SA) node pacemaker cells. The atrium is simulated by a two-dimensional array of pacemaker cells (25 × 25), composed of a region of SA node pacemaker cells (11 × 11) surrounded by atrial pacemaker cells. All pacemakers cells are characterized by only the most relevant functional properties, those which play the most direct role in the determination of the cardiac rate and in the mechanism of arrhythmias. These properties are: the intrinsic cycle length, τ, an `internal' feature of each pacemaker cell, and the phase-response curve (PRC), an `overall collective' function. The PRC embodies the interactions of each pacemaker cell with its neighboring cells, and thus represents the type of connection (strong, weak, etc.) of the pacemaker cell with its surroundings. In our model, the SA node region differs from the atrial region by cycle length distribution and PRCs. We studied the spatial interaction between SA node pacemaker cells and atrial pacemaker cells as a function of the regional variation of cells properties and as a function of the “electrical” coupling between cells (the PRC), in the SA node region, in the atrial region, and in a border zone between them. We investigated the influence of those parameters on the activation pattern, on the conduction time of the array, and on a pseudo-ECG signal. This study demonstrates that by representing the atrial cells as a population of `pacemaker-like' cells, similar to the SA node pacemaker cells, but differing markedly in their cycle lengths and cell-to-cell interaction (PRC), we can create a global picture of the atrial system by applying a simple physical-mathematical model. This approach enables us to explore physiological phenomena related to the genesis and maintenance of atrial activity. It also reveals the conditions which predispose to atrial arrhythmias and conduction disturbances (e.g. tachycardia, pacemaker shift, re-entry, fibrillation). In particular, it yields insight into the mechanism of transition from normal atrial activity to the disordered state of atrial fibrillation. Therefore, this study suggests a new way of looking at the development of cardiac arrhythmias of atrial origin. Received: 8 September 1997 / Accepted in revised form: 6 October 1998     

Mathematical models of action potentials in the periphery and center of the rabbit sinoatrial node

Mathematical models of the action potential in the periphery and center of the rabbit sinoatrial (SA) node have been developed on the basis of published experimental data. Simulated action potentials are consistent with those recorded experimentally: the model-generated peripheral action potential has a more negative takeoff potential, faster upstroke, more positive peak value, prominent phase 1 repolarization, greater amplitude, shorter duration, and more negative maximum diastolic potential than the model-generated central action potential. In addition, the model peripheral cell shows faster pacemaking. The models behave qualitatively the same as tissue from the periphery and center of the SA node in response to block of tetrodotoxin-sensitive Na(+) current, L- and T-type Ca(2+) currents, 4-aminopyridine-sensitive transient outward current, rapid and slow delayed rectifying K(+) currents, and hyperpolarization-activated current. A one-dimensional model of a string of SA node tissue, incorporating regional heterogeneity, coupled to a string of atrial tissue has been constructed to simulate the behavior of the intact SA node. In the one-dimensional model, the spontaneous action potential initiated in the center propagates to the periphery at approximately 0.06 m/s and then into the atrial muscle at 0.62 m/s.     

Parasympathetic control of the heart. II. A novel interganglionic intrinsic cardiac circuit mediates neural control of heart rate.

Intracardiac pathways mediating the parasympathetic control of various cardiac functions are incompletely understood. Several intracardiac ganglia have been demonstrated to potently influence cardiac rate [the sinoatrial (SA) ganglion], atrioventricular (AV) conduction (the AV ganglion), or left ventricular contractility (the cranioventricular ganglion). However, there are numerous ganglia found throughout the heart whose functions are poorly characterized. One such ganglion, the posterior atrial (PA) ganglion, is found in a fat pad on the rostral dorsal surface of the right atrium. We have investigated the potential impact of this ganglion on cardiac rate and AV conduction. We report that microinjections of a ganglionic blocker into the PA ganglion significantly attenuates the negative chronotropic effects of vagal stimulation without significantly influencing negative dromotropic effects. Because prior evidence indicates that the PA ganglion does not project to the SA node, we neuroanatomically tested the hypothesis that the PA ganglion mediates its effect on cardiac rate through an interganglionic projection to the SA ganglion. Subsequent to microinjections of the retrograde tracer fast blue into the SA ganglion, >70% of the retrogradely labeled neurons found within five intracardiac ganglia throughout the heart were observed in the PA ganglion. The neuroanatomic data further indicate that intraganglionic neuronal circuits are found within the SA ganglion. The present data support the hypothesis that two interacting cardiac centers, i.e., the SA and PA ganglia, mediate the peripheral parasympathetic control of cardiac rate. These data further support the emerging concept of an intrinsic cardiac nervous system.     

Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border.

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.     

Effects of external stimuli on the pacemaker function of the sinoatrial node in sodium channel gene mutations models

Loss of function and gain of function mutations of the sodium channel were investigated using an intact two-dimensional rabbit sinoatrial node (SAN) and atrial cell model. The effects of three external stimuli (acetylcholine secretion by the vagal nerve, acid-base concentration, and tissue temperature) on cardiac pacemaker function and conduction were studied. Our results show that these two groups of mutations have different effects on pacemaker function and conduction. Furthermore, we found that the negative effects of these mutations could be altered by external stimuli. The bradycardic effects of mutations were magnified by an increase in acetylcholine level. Changes in acid-base concentration and tissue temperature increased the ability of the SAN to recover its pacemaker function. The results of this study increase our understanding of sodium channel disorders, and help to advance research on the treatment of these conditions.     

Changes in conduction within the diffuse sinoatrial node of the chicken following premature atrial stimulation

Premature atrial stimulation was used to estimate sinoatrial conduction within the diffuse sinoatrial node of the bird (chicken), and compare its conduction with that reported for mammals. While sinoatrial conduction could not be determined in the chicken because reset did not occur, the premature wavefront did have an effect on the sinoatrial node because the recovery interval following the premature stimulus became less than compensatory with shortening of the premature stimulus interval. This less than compensatory non-reset recovery interval is interpreted as a conduction dependent response in which the intrinsic wavefront leading to the first recovery atrial activation conducts out of the node faster than normal. This conduction dependent recovery interval is seen infrequently in mammals (rabbit, dog and man). The absence of reset and the presence of a less than compensatory non-reset response in the chicken suggests that while the general organization of the sinoatrial node of the chicken is similar to that in mammals, a larger transitional cell network in the chicken prevents a premature wavefront from reaching the pacemaker cells and resetting them.     

Beating irregularity of single pacemaker cells isolated from the rabbit sinoatrial node.

Single pacemaker heart cells discharge irregularly. Data on fluctuations in interbeat interval of single pacemaker cells isolated from the rabbit sinoatrial node are presented. The coefficient of variation of the interbeat interval is quite small, approximately 2%, even though the coefficient of variation of diastolic depolarization rate is approximately 15%. It has been hypothesized that random fluctuations in interbeat interval arise from the stochastic behavior of the membrane ionic channels. To test this hypothesis, we constructed a single channel model of a single pacemaker cell isolated from the rabbit sinoatrial node, i.e., a model into which the stochastic open-close kinetics of the individual membrane ionic channels are incorporated. Single channel conductances as well as single channel open and closed lifetimes are based on experimental data from whole cell and single channel experiments that have been published in the past decade. Fluctuations in action potential parameters of the model cell are compared with those observed experimentally. It is concluded that fluctuations in interbeat interval of single sinoatrial node pacemaker cells indeed are due to the stochastic open-close kinetics of the membrane ionic channels.     

Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node

We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (If or Ih) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brain If cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1-3. The transmembrane region of HAC4 was most homologous to partially cloned mouse If BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at -87.2 +/- 2.8 mV under control conditions and at -64.4 +/- 2.6 mV in the presence of intracellular 0.3 mM cAMP. The reversal potential was -34.2 +/- 0.9 mV in 140 mM Na+o and 5 mM K+o versus 10 mM Na+i and 145 mM K+i. These results indicate that HAC4 forms If in rabbit heart SA node.     

Current developments on the physiology of the cardiac atrial pacemaker an excitation conduction of the left atrium]

According to recent developments the atrial pacemaker area and the right atrium show a peculiar morpho-functional organization, i.e.: 1) The pacemaker area is formed of clusters of cells containing relatively few myofibrils and showing embryologic characteristics. Such cells are known as nodal cells and between these and the atrial muscles are in general situated transitional cells. Each cluster is separated from the other by collagenous boundaries. The resistance of the membranes to the current flow seems to be relatively low between the cells of the same cluster but the collagenous boundaries are, according to TRAUTWEIN e UCHIZONO (1963), very poor conductors. The pacemaker activity seems to originate inside the various clusters. 2) The functional relationships between the sinoatrial node and the atrioventricular node as well as the interatrial relationship would take place through preferential pathways. These pathways corresponding approximately to the tracts described by JAMES (1966) (anterior, posterior and middle internodal tracts) and to the interatrial or Bachmann bundle, seems to show a higher velocity conduction. In general the fibres of which the tracts are composed are neither morphologically nor functionally isolated from the atrial muscle. The functional consequences of the above mentioned nodal and atrial organization seems to be: a) The possible conditioning of the pacemaker functions by the various clusters activity i.e. the dominance of one cluster over another. b) The shifting of the pacemaker activity from one cluster to anothr due to the arrival of nervous stimuli or chemical substances, etc. According to some Authors as a consequence of the shift the pacemaker area can sometimes move out side the nodal tissue and settle inside an area belonging to the internodal pathways. c) Another consequence of the shift can be the different involvement of the conducting pathways which can lead to a change in the dynamics of the atrial invasion by the excitement.     

Expression of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heart development

HCN4 is a hyperpolarization-activated nucleotide-gated cation channel involved in the generation of the I(f) current that drives cardiac pacemaker activity. Previous studies have demonstrated that HCN4 is highly expressed in a restricted manner in adult sinoatrial (SA) node [Eur. J. Biochem. 268 (2001) 1646]. However, its developmental expression pattern is unknown. We have examined expression of HCN4 mRNA during mouse heart development. HCN4 mRNA was first detected in the cardiac crescent at embryonic day (ED) 7.5. At ED 8 it was symmetrically located in the most caudal portion of the heart tube, the sinus venosus where pacemaker activity has previously been reported [Am. J. Physiol. 212 (1967) 407]. With further development, HCN4 expression became asymmetrically distributed, occupying the dorsal wall of the right atria, and was progressively restricted to the junction of the right atrial appendage and the superior vena cava. The site of HCN4 expression in late embryonic heart coincided with the location of the SA node in postnatal and adult heart [Cardiovasc. Res. 52 (2001) 51]. Our results suggest that HCN4 may be a unique marker of the developing SA node.     

Gap junctions in the rabbit sinoatrial node

In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels.     

Presence of the Kv1.5 K(+) channel in the sinoatrial node.

The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K(+) channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, approximately 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions.