Computer simulations of pacemaker shift in the sinoatrial node

The initiation and propagation of electrical pulses in the sinoatrial node under normal conditions and after application of acetylcholine have been simulated. It has been found that normally a single or a few leading centers are formed in the tissue. When acetylcholine is applied, a temporary functional block of conduction may appear; the leading center migrates under these conditions.     

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.     

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.     

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.     

Electrophysiology and pacemaker function of the developing sinoatrial node

The sinoatrial node performs its task as a cardiac impulse generator throughout the life of the organism, but this important function is not a constant. Rather, there are significant developmental changes in the expression and function of ion channels and other cellular elements, which lead to a postnatal slowing of heart rate and may be crucial to the reliable functioning of the node during maturation. In this review, we provide an overview of current knowledge regarding these changes, with the main focus placed on maturation of the ion channel expression profile. Studies on Na(+) and pacemaker currents have shown that their contribution to automaticity is greater in the newborn than in the adult, but this age-dependent decrease is at least partially opposed by an increased contribution of L-type Ca(2+) current. Whereas information regarding age-dependent changes in other transmembrane currents within the sinoatrial node are lacking, there are data on other relevant parameters. These include an increase in the nodal content of fibroblasts and in the area of nonexpression of connexin43, considered a molecular marker of nodal tissue. Although much remains to be done before a comprehensive view of the developmental biology of the node is available, important evidence in support of a molecular interpretation of developmental slowing of the intrinsic sinoatrial rate is beginning to emerge.     

Role of dual pacemaker mechanisms in sinoatrial node discharge

We investigated whether in the sinoatrial node (SAN) there are two different pacemaker mechanisms and whether either one can maintain spontaneous discharge. These questions were studied by means of an electrophysiological technique and of blockers of different diastolic currents in rabbit and guinea pig isolated SAN. In SAN subsidiary pacemakers of both species, Cs(+) (5-10 mM) or high [K(+)](o) (10-12 mM) decreased the maximum diastolic potential, abolished diastolic depolarization (DD) at polarized levels (subsidiary DD), unmasked a U-shaped dominant DD at depolarized levels, but did not stop the SAN. In rabbit SAN, E4031 (1 microM) and d-sotalol (100 microM) did not stop discharge, but did so after block of subsidiary DD by high [K(+)](o) or Cs(+). In guinea pig SAN, in Tyrode solution E4031, d-sotalol or indapamide (100 microM) did not stop SAN discharge. In the presence of Cs(+) or high [K(+)](o) indapamide (but not E4031 or d-sotalol) stopped the SAN. Ba(2+) (1-5 mM) led to stoppage of discharge both in Tyrode solution and in high [K(+)](o) or Cs(+). Depolarization by blockers of DD unmasked sinusoidal fluctuations, which during recovery were responsible for resumption of discharge. We conclude that in rabbit and guinea pig SAN, two different pacemaker mechanisms (Cs(+)- and K(+)-sensitive subsidiary DD, and Cs(+)- and K(+)-insensitive dominant DD) can independently sustain discharge, but block of both mechanisms leads to quiescence. Abolition of dominant DD by blockers of I(K) is consistent with a decay of I(K) as the dominant pacemaking mechanism, I(Kr) being more important in rabbit and I(Ks) in guinea pig. Sinusoidal fluctuations appear to be an essential component of the pacemaking process.     

Ultrastructure of the pacemaker cells of the sinoatrial node in rats

Ultrastructure of the cells generating the action potential, specific for the pacemaker of the sinuous-auricular node has been studied. The cells are labelled with lanthanum chloride by means of the registrating microelectrode. Two types of pacemakers are revealed. The cells of one type contain specific auricular granules, while those of the other type do not contain them. The pacemaker-cells of the sinuous-auricular node have some peculiarities in the structure of the contractile apparatus, mitochondria, Golgi complex, intercellular contacts owing to which their morphological identification is possible.     

Computer simulation of the preautomatic pause in pacemaker cells of the sinoatrial node

The duration of the preautomatic pause as a function of sinoatrial node, the type of pacemaker cells, acetylcholine concentration, the duration of high-frequency stimulation, and the conductivity of gap junctions has been studied. It was found that the preautomatic pause in peripheral pacemakers occurs at a higher concentration of acetylcholine as compared with central pacemakers. The dependence of the duration of the preautomatic pause on the gap junction conductivity is a nonlinear one.     

The mechanism of setting the common rhythm of the pacemaker pair in the sinoatrial node

Principal physiological hypotheses concerning the setting of united rhythm in the heart sinoatrial node (SAN) are considered. A mathematical model of SAN is proposed which takes into account properties of individual elementary pacemakers and their interaction. Assuming paired interaction of the pacemakers there are revealed the main P.D. parameters, affecting the setting of the united rhythm. Quantitative expressions are obtained for the united rhythm period, delay and propagation velocity of the excitation. The calculated data are compared with the experimental ones. The hypothesis concerning the setting of the united rhythm as a result of the interaction of SAN pacemakers is confirmed.     

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.     

The end effector of circadian heart rate variation: the sinoatrial node pacemaker cell

Cardiovascular function is regulated by the rhythmicity of circadian, infradian and ultradian clocks. Specific time scales of different cell types drive their functions: circadian gene regulation at hours scale, activation-inactivation cycles of ion channels at millisecond scales, the heart''s beating rate at hundreds of millisecond scales, and low frequency autonomic signaling at cycles of tens of seconds. Heart rate and rhythm are modulated by a hierarchical clock system: autonomic signaling from the brain releases neurotransmitters from the vagus and sympathetic nerves to the heart’s pacemaker cells and activate receptors on the cell. These receptors activating ultradian clock functions embedded within pacemaker cells include sarcoplasmic reticulum rhythmic spontaneous Ca²⁺ cycling, rhythmic ion channel current activation and inactivation, and rhythmic oscillatory mitochondria ATP production. Here we summarize the evidence that intrinsic pacemaker cell mechanisms are the end effector of the hierarchical brain-heart circadian clock system. [BMB Reports 2015; 48(12): 677-684]     

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.     

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.     

Investigation of the synchronization of the sinoatrial node upon stimulation from atria

The excitation of the sinoatrial node from frog heart atria has been experimentally investigated. Potentials were measured by means of microelectrodes introduced in pacemaker cells of the sinoatrial node. It has been found that atria can modulate the rhythm of the sinoatrial node due to electric and electromechanical actions, among which the electromechanical action is more important. Specific transient processes accompanying the establishment of the stationary rhythm have been studied. A mathematical model of the transient processes of achieving the rhythm of the sinoatrial node is proposed on the basis of Diophantine methods. The calculations performed using the mathematical model satisfactorily agree with the experimental results. The stabilizing role of atria in forming the rhythm of the sinoatrial node is revealed.     

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.     

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.