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.     

Pacemaker activity of the rabbit sinoatrial node. A comparison of mathematical models.

In the past decade, three mathematical models describing the pacemaker activity of the rabbit sinoatrial node have been developed: the Bristow-Clark model, the Irisawa-Noma model, and the Noble-Noble model. In a comparative study it is demonstrated that these models, as well as subsequent modifications, all have several drawbacks. A more accurate model, describing the pacemaker activity of a single pacemaker cell isolated from the rabbit sinoatrial node, was constructed. Model equations, including equations for the T-type calcium current, are based on experimental data from voltage clamp experiments on single cells that were published during the last few years. In contrast to the other models, only a small amount of background current contributes to the overall electrical charge flow. The action potential parameters of the model cell, its responses to voltage clamp steps and its current-voltage relationships have been computed. The model is used to discuss the relative contribution of membrane current components to the slow diastolic depolarization phase of the action potential.     

A mathematical model of action potentials of mouse sinoatrial node cells with molecular bases

Genetically modified mice are popular experimental models for studying the molecular bases and mechanisms of cardiac arrhythmia. A postgenome challenge is to classify the functional roles of genes in cardiac function. To unveil the functional role of various genetic isoforms of ion channels in generating cardiac pacemaking action potentials (APs), a mathematical model for spontaneous APs of mouse sinoatrial node (SAN) cells was developed. The model takes into account the biophysical properties of membrane ionic currents and intracellular mechanisms contributing to spontaneous mouse SAN APs. The model was validated by its ability to reproduce the physiological exceptionally short APs and high pacing rates of mouse SAN cells. The functional roles of individual membrane currents were evaluated by blocking their coding channels. The roles of intracellular Ca(2+)-handling mechanisms on cardiac pacemaking were also investigated in the model. The robustness of model pacemaking behavior was evaluated by means of one- and two-parameter analyses in wide parameter value ranges. This model provides a predictive tool for cellular level outcomes of electrophysiological experiments. It forms the basis for future model development and further studies into complex pacemaking mechanisms as more quantitative experimental data become available.     

Mechanism of outercellular [K+] in generation of pacemaker action potentials in sinoatrial valve of the rabbit

In a control solution (solution I; 3 mM K+, 35 degrees C) the amplitude of APs of cells in the valve centre was 81 +/- 6 mV (Mean +/- SEM), the maximal upstroke velocity (dV/dtmax) was 19 +/- 4 V/s, velocity slow diastolic depolarization (DD) - 65 +/- 8 mV/s, the rate of spontaneous AP generation was 135 +/- 14 bpm. Hypo K+ (50 %) solution (solution II) decreased slow DD by - 22 % and dV/dtmax by -58 % and the rate of AP generation by 15 % compared to the control solution. In saline solution, decrease of Emax from -68 to -45 mV and four-fold decrease of slow DD and dV/dtmax were registered at the 8th of exposure. After that the Emax noise and origin of early (EADs) and delay (DADs) afterdepolarizations were observed. Comparative analysis of the main AP parameters of SA valve latent pacemaker cells in solution III (without K+) and solution IV (without KC1 and CaCl2) exposure demonstrated that changes of this parameters in solution IV were less then in solution III. It is interesting that in solution IV exposure the phase of the early repolarization (phase 1) was differentiated, dV/dtmax was increased by 21%. The frequency of AP generation decreased by 18 % as in solution with 50% K+. At the same time, EADs were not registered. It seems that the decreasing of the transsarcolemal gradient of K+ depolarized Emax despite Nernst equation. At the same time permeability was decreased for K+ and Na+ ions. That could involve Ca2+ overload and origin of EADs and DADs.     

Effects of streptozotocin-induced diabetes on action potentials in the sinoatrial node compared with other regions of the rat heart

In vivo biotelemetry studies have demonstrated that heart rate (HR) is progressively and rapidly reduced after administration of streptozotocin (STZ) and that the reduction in HR can be partially normalized with insulin replacement. Reductions in HR have also been reported in isolated perfused heart and superfused right atrial preparations suggesting that intrinsic defects in the heart are at least partly responsible for the bradycardia. The regional effects of STZ-induced diabetes mellitus (DM) on action potentials (APs) in the sinoatrial node (SAN), right and left atria and ventricles have been compared in the spontaneously beating Langendorff perfused rat heart 10–12 weeks after treatment. HR was significantly reduced in STZ-induced diabetic rat heart (174 ± 9 BPM) compared to controls (241 ± 12 BPM). The duration of AP repolarization at 50% and 70% from peak AP was significantly prolonged in SAN, right atrium and right ventricle from STZ-induced diabetic rat compared to age-matched controls. In the SAN AP duration (APD) at 50% and 70% were 51.7 ± 2.2 and 59.5 ± 2.3 ms in diabetic rat heart compared to 45.2 ± 1.7 and 50.0 ± 1.6 ms in controls, respectively. In contrast APD at 50% and 70% were not significantly altered in the left atrium and left ventricle. Regional defects in the expression and/or electrophysiology of SAN ion channels, and in particular those involved in AP repolarization, might underlie heart rhythm disturbances in the STZ-induced DM rat.     

Morphological study of the innervation pattern of the rabbit sinoatrial node

The pattern of nerves, ganglia, and fine nerve processes in the adult rabbit sinoatrial node, identified by microelectrode recording, was defined by staining histochemically for cholinesterase followed by silver impregnation. A generalized repeatable pattern of innervation was recognized, including 1) a large ganglionic complex inferior to the sinoatrial node; 2) two or three moderately large nerves traversing the sinoatrial node parallel to the crista terminalis; 3) nerves entering the region from the atrial septum, the superior vena cava, and the inferior vena cava; and 4) a fine network of nerve processes, particularly extensive in the morphologically dense small-cell part of the sinoatrial node. When the site of initial depolarization in the node was located and marked by a broken-off electrode tip, it was found, after cholinesterase staining, to be characterized by a cluster of cells enclosed in a nest or basket of fine nerves. Similar nested cell clusters were observed elsewhere in the sinoatrial node in this same preparation and in other hearts. A complex interweaving of atrial muscle fibers was observed medial and inferomedial to the sinoatrial node, which may form the anatomical basis for the lack of conduction through this region. The morphological pattern of nerves, ganglia, and myocardial cells described in this study emphasizes the complexity of innervation of the sinoatrial node, including its intrinsic neural elements. Cholinesterase/silver staining can be useful in the definition and comparison of electrophysiologically identified sites within the sinoatrial node.     

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.     

Acetylcholine inhibition in rabbit sinoatrial node is prevented by pertussis toxin

The effects of acetylcholine (ACh) were examined on the naturally occurring slow action potentials (APs) of the isolated, organ-cultured, spontaneously beating sinoatrial (SA) node of the rabbit, in the presence or absence of pertussis toxin. The sensitivity of the SA-node preparations to ACh was not altered after 24 h incubation in organ culture medium. Activation of the muscarinic receptor hyperpolarized the cells and reduced the frequency of spontaneous activity at low concentrations (1 X 10(-6) and 3 X 10(-6) M), and completely abolished automaticity at higher concentrations (1 X 10(-5) M). However, stimulated activity was maintained. Increased concentrations (1 X 10(-4) M) of ACh completely abolished excitability. When the SA-node preparations were cultured in the presence of 0.5 micrograms/mL pertussis toxin, concentrations of ACh as high as 1 X 10(-4) M had no effect on the AP parameters and frequency of spontaneous activity. The results indicate that inactivation of G proteins by pertussis toxin caused inhibition of the ACh effects on the automaticity of the SA node. In addition, the blocking effect of ACh to the naturally occurring slow APs was also inhibited by pertussis toxin. We conclude that in the rabbit SA node, the effects of ACh on automaticity and on the slow channels are mediated by G protein.     

Investigation of pacemaker shift in the rabbit sinoatrial node using the optical mapping technique

Changes of the activation sequence in the rabbit sinoatrial node under the influence of low temperature and I _f selective blocker ivabradine have been studied using the optical mapping technique. Both factors caused a shift of the pacemaker within the sinoatrial node region. These results are compared with the data obtained recently in the investigation of pacemaker shift under the influence of cholinergic and adrenergic factors. Possible mechanisms of the pacemaker shift are discussed. The suppression of electric activity in the central part of the sinoatrial node during the action of acetylcholine, which is called cholinergic inexcitability, may be considered as one of the mechanisms of the pacemaker shift. It is shown that the main cause of cholinergic inexcitability is the activation of potassium acetylcholine-dependent current I _KACh.     

Postganglionic nerve stimulation induces temporal inhibition of excitability in rabbit sinoatrial node

Vagal stimulation results in complex changes of pacemaker excitability in the sinoatrial node (SAN). To investigate the vagal effects in the rabbit SAN, we used optical mapping, which is the only technology that allows resolving simultaneous changes in the activation pattern and action potentials morphologies. With the use of immunolabeling, we identified the SAN as a neurofilament 160-positive but connexin 43-negative region (n = 5). Normal excitation originated in the SAN center with a cycle length (CL) of 405 +/- 14 ms (n = 14), spread anisotropically along the crista terminalis (CT), and failed to conduct toward the septum. Postganglionic nerve stimulation (PNS, 400-800 ms) reduced CL by 74 +/- 7% transiently and shifted the leading pacemaker inferiorly (78%) or superiorly (22%) from the SAN center by 2-10 mm. In the intercaval region between the SAN center and the septal block zone, PNS produced an 8 +/- 1-mm(2) region of transient hyperpolarization and inexcitability. The first spontaneous or paced excitation following PNS could not enter this region for 500-1,500 ms. Immunolabeling revealed that the PNS-induced inexcitable region is located between the SAN center and the block zone and has a 2.5-fold higher density of choline acetyltransferase than CT but is threefold lower than the SAN center. The fact that the inexcitability region does not coincide with the most innervated area indicates that the properties of the myocytes themselves, as well as intercellular coupling, must play a role in the inexcitability induction. Optically mapping revealed that PNS resulted in transient loss of pacemaker cell excitability and unidirectional entrance conduction block in the periphery of SAN.     

降钙素基因相关肽对家兔离体窦房结电生理活动的影响

用常规微电极方法研究了降钙素基因相关肽（ＣＧＲＰ）对家兔窦房结起搏细胞的电生理作用,并进一步探讨这种作用与钙电流的关系。结果：⑴低浓度ＣＧＲＰ（１ｎｍｏｌ／Ｌ）对窦房结动作电位各参数无显著影响;中等浓度ＣＧＲＰ（１０ｎｍｏｌ／Ｌ）可增加最大舒张期电位、动作电位幅度、０期最大除极化速率和４期自动除极速率,缩短窦性周期、动作电位复极化５０％和９０％时间,这些作用经２０ｍｉｎ达到高峰;高浓度ＣＧＲＰ（２     

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.     

Telocytes in the human sinoatrial node

The sinoatrial node (SAN) is composed mostly of pacemaker, transitional and Purkinje‐like cells. Pacemaker cells, especially in the centre of the SAN, are surrounded by dense fibrous tissue and do not have any contact with transitional cells. We hypothesize that the SAN contains telocytes that have contacts with pacemaker cells and contractile myocardium. Immunohistochemistry using antibodies against HCN4 and antibody combinations against CD34 and HCN4 was carried out on 12 specimens. Confocal laser scanning microscopy (CLSM) with two mixtures of primary antibodies, namely CD34/S100 and vimentin/S100, was performed in three cases. In two cases, CLSM was carried out with CD117 antibody. Specimens for electron microscopy and immunocytochemistry with HCN4 immunogold labelling were taken from another three patients. In our study, we found cells with the immunophenotype of telocytes in the SAN. There were twice as many of these cells in the centre of the SAN as in the periphery (20.3 ± 4.8 versus 10.8 ± 4.4 per high‐power field). They had close contact with pacemaker cells and contractile cardiomyocytes and expressed HCN4. The ultrastructural characteristics of these cells are identical to those of telocytes observed earlier in other organs. Our study provides evidence that telocytes are present in the SAN.     

Direction-selective dendritic action potentials in rabbit retina

Dendritic spikes that propagate toward the soma are well documented, but their physiological role remains uncertain. Our in vitro patch-clamp recordings and two-photon calcium imaging show that direction-selective retinal ganglion cells (DSGCs) utilize orthograde dendritic spikes during physiological activity. DSGCs signal the direction of image motion. Excitatory subthreshold postsynaptic potentials are observed in DSGCs for motion in all directions and provide a weakly tuned directional signal. However, spikes are generated over only a narrow range of motion angles, indicating that spike generation greatly enhances directional tuning. Our results indicate that spikes are initiated at multiple sites within the dendritic arbors of DSGCs and that each dendritic spike initiates a somatic spike. We propose that dendritic spike failure, produced by local inhibitory inputs, might be a critical factor that enhances directional tuning of somatic spikes.     

Rhythmogenesis in the heart sinoatrial node

The most significant experimental data on the formation of the common rhythm of the heart sinoatrial node are presented for both the intact heart sinoatrial node and cardiomyocytes in cell structures. The basic mathematical models for studying the synchronization processes in the sinoatrial node, including the Noble equation, Bonhoffer-van der Pol model, and modified axiomatic models, are described. The basic results obtained with the mathematical models are presented. The most important causes affecting the formation of the common rhythm—the pacemaker potential shape in the slow diastolic depolarization phase, its porosity, the coupling force between pacemakers, and the electrical power of pacemakers—are revealed. Rhythmogenesis is studied using the modified axiomatic model. The method allows the calculation of the common rhythm of the sinoatrial node, with allowance for the mutual effect of the pacemaker cells, including the coupling force, electric power of cells, and possibility of the cells clustering. It has been shown that the common rhythm of the sinoatrial node is generally formed at the intermediate level of the rhythms of all pacemaker cells.     

Ischemia alters the electrical activity of pacemaker cells isolated from the rabbit sinoatrial node

The purpose of this study was to investigate the mechanisms responsible for ischemia-induced changes in spontaneous electrical activity. An ischemic-like Tyrode solution (pH 6.6) reversibly depolarized the maximum diastolic potential (MDP) and reduced the action potential (AP) overshoot (OS). We used SNARF-1, which is an indicator of intracellular pH (pH(i)), and perforated-patch techniques to test the hypothesis that acidosis caused these effects. Acidic but otherwise normal Tyrode solution (pH 6.8) produced similar effects. Basic Tyrode solution (pH 8.5) hyperpolarized the MDP, shortened the AP, and slowed the firing rate. In the presence of "ischemic" Tyrode solution, hyperpolarizing current restored the MDP and OS to control values. HOE-642, an inhibitor of Na/H exchange, did not alter pH(i) or electrical activity and did not prevent the effects of ischemic Tyrode solution or recovery after washout. Time-independent net inward current but not hyperpolarization-activated inward current was enhanced by ischemic Tyrode solution or by 30 microM BaCl(2), a selective blocker of inward-rectifying K currents at this concentration. The results suggest that 1) acidosis was responsible for the ischemia-induced effects but Na/H exchange was not involved, 2) the OS was reduced because of depolarization-induced inactivation of inward currents that generate the AP upstroke, and 3) reduction of an inward-rectifying outward K current contributed to the depolarization.     

Simulated ischemia enhances L-type calcium current in pacemaker cells isolated from the rabbit sinoatrial node

Ischemic-like conditions (a glucose-free, pH 6.6 Tyrode solution bubbled with 100% N(2)) enhance L-type Ca current (I(Ca,L)) in single pacemaker cells (PCs) isolated from the rabbit sinoatrial node (SAN). In contrast, studies of ventricular myocytes have shown that acidic extracellular pH, as employed in our "ischemic" Tyrode, reduces I(Ca,L). Therefore, our goal was to explain why I(Ca,L) is increased by "ischemia" in SAN PCs. The major findings were the following: 1) blockade of Ca-induced Ca release with ryanodine, exposure of PCs to BAPTA-AM, or replacement of extracellular Ca(2+) with Ba(2+) failed to prevent the ischemia-induced enhancement of I(Ca,L); 2) inhibition of protein kinase A with H-89, or calcium/calmodulin-dependent protein kinase II with KN-93, reduced I(Ca,L) but did not prevent its augmentation by ischemia; 3) ischemic Tyrode or pH 6.6 Tyrode shifted the steady-state inactivation curve in the positive direction, thereby reducing inactivation; 4) ischemic Tyrode increased the maximum conductance but did not affect the activation curve; 5) in rabbit atrial myocytes isolated and studied with exactly the same techniques used for SAN PCs, ischemic Tyrode reduced the maximum conductance and shifted the activation curve in the positive direction; pH 6.6 Tyrode also shifted the steady-state inactivation curve in the positive direction. We conclude that the acidic pH of ischemic Tyrode enhances I(Ca,L) in SAN PCs, because it increases the maximum conductance and reduces inactivation. Furthermore, the opposite results obtained with rabbit atrial myocytes cannot be explained by differences in cell isolation or patch-clamp techniques.