Bimodal delta-opioid receptors regulate vagal bradycardia in canine sinoatrial node

Methionine-enkephalin-arginine-phenylalanine (MEAP) introduced into the interstitium of the canine sinoatrial (SA) node by microdialysis interrupts vagal bradycardia. In contrast, raising endogenous MEAP by occluding the SA node artery improves vagal bradycardia. Both are blocked by the same delta-selective antagonist, naltrindole. We tested the hypothesis that vagal responses to intranodal enkephalin are bimodal and that the polarity of the response is both dose- and opioid receptor subtype dependent. Ultralow doses of MEAP were introduced into the canine SA node by microdialysis. Heart rate frequency responses were constructed by stimulating the right vagus nerve at 1, 2, and 3 Hz. Ultralow MEAP infusions produced a 50-100% increase in bradycardia during vagal stimulation. Maximal improvement was observed at a dose rate of 500 fmol/min with an ED50 near 50 fmol/min. Vagal improvement was returned to control when MEAP was combined with the delta-antagonist naltrindole. The dose of naltrindole (500 fmol/min) was previously determined as ineffective vs. the vagolytic effect of higher dose MEAP. When MEAP was later reintroduced in the same animals at nanomoles per minute, a clear vagolytic response was observed. The delta1-selective antagonist 7-benzylidenenaltrexone (BNTX) reversed the vagal improvement with an ED50 near 1 x 10-21 mol/min, whereas the delta2-antagonist naltriben had no effect through 10-9 mol/min. Finally, the improved vagal bradycardia previously associated with nodal artery occlusion and endogenous MEAP was blocked by the selective delta1-antagonist BNTX. These data support the hypothesis that opioid effects within the SA node are bimodal in character, that low doses are vagotonic, acting on delta1-receptors, and that higher doses are vagolytic, acting on delta2-receptors.     

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.     

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.     

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.     

Computer simulation of microreentry in the sinoatrial node

We have studied the dynamics of reentry inside the sinoatrial node (SAN). We have found that reentry is unstable at high intercellular conductance. Rotating reentry induces a slow migrating crescent-shaped functional block near the SAN boundary. Abnormal conduction from atrial tissue into the SAN occurs after decay of the reentry. Acetylcholine increases the lifespan of reentry in the SAN.     

Cardiac enkephalins attenuate vagal bradycardia: interactions with NOS-1-cGMP systems in canine sinoatrial node

Endogenous opioids and nitric oxide (NO) are recognized modulators of cardiac function. Enkephalins and inhibitors of NO synthase (NOS) both produce similar interruptions in the vagal control of heart rate. This study was conducted to test the hypothesis that NO systems within the canine sinoatrial (SA) node facilitate local vagal transmission and that the endogenous enkephalin methionine-enkephalin-arginine-phenylalanine (MEAP) attenuates vagal bradycardia by interrupting the NOS-cGMP pathway. Microdialysis probes were inserted into the SA node, and they were perfused with nonselective (Nomega-nitro-l-arginine methyl ester) and neuronal (7-nitroindazole) NOS inhibitors. The right vagus nerve was stimulated and both inhibitors gradually attenuated the resulting vagal bradycardia. The specificity of these inhibitions was verified by an equally gradual reversal of the inhibition with an excess of the NOS substrate l-arginine. Introduction of MEAP into the nodal interstitium produced a quickly developing but quantitatively similar interruption of vagal bradycardia that was also slowly reversed by the addition of l-arginine and not by d-arginine. Additional support for convergence of opioid and NO pathways was provided when the vagolytic effects of MEAP were also reversed by the addition of the NO donor S-nitroso-N-acetyl-penicillamine, the protein kinase G activator 8-bromo-cGMP, or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. MEAP and 7-nitroindazole were individually combined with the direct acting muscarinic agonist methacholine to evaluate potential interactions with muscarinic receptors within the SA node. MEAP and 7-nitroindazole were unable to overcome the bradycardia produced by methacholine. These data suggest that NO and enkephalins moderate the vagal control of heart rate via interaction with converging systems that involve the regulation of cAMP within nodal parasympathetic nerve terminals.     

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.     

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.     

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.     

Verapamil potentiates vagally mediated sinoatrial chronotropic responses in dogs

A brief burst of electrical stimuli delivered to the vagus nerve during the cardiac cycle elicits a triphasic cardiac chronotropic response. The cardiac cycle length initially increases, then briefly decreases, and subsequently increases again. We studied the effects of a calcium channel blocking agent, verapamil, on these responses to vagal stimulation during sinoatrial nodal rhythm in anesthetized, open-chest dogs. Verapamil increased the basal cardiac cycle length only slightly; however, the primary cardioinhibition was accentuated approximately 40% (from 396 to 555 ms) by verapamil. Neither the acceleratory phase of this triphasic response nor the secondary cardioinhibition was significantly affected by verapamil. These results indicate that verapamil potentiates the initial action of acetylcholine at the sinoatrial node when the vagus is activated with brief stimuli.     

To the formation of a unified rhythm in the heart sinoatrial node

The dynamics of establishing a unified sinoatrial node rhythm are considered. Mutual synchronization is shown to result in phase shifts and excitation delays. Rhythmogenesis in systems of two or many interacting pacemaker cells is examined in several point models and distributed models (Noble, Bonhoeffer-van der Pol, FitzHugh, Hodgkin-Huxley, Morris-Lecar).     

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.     

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.     

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.     

Anatomy of the sinoatrial node and the supply of its vascularization in man

Seventy heart preparations of persons belonging to different sex and age have been investigated, using a complex of anatomical and histological techniques. The dimensions of the sinoatrial node (SAN) vary with age and depend on various size and form of the heart. The large atrial branch of the right and left coronary arteries supplies mainly the SAN with blood. More seldom the atrial branches of both cardiac arteries, having anastomoses, realize the SAN blood supply. The character of the SAN vascularization depends on branching variations of the atrial vessels. At the right coronary variant the sources of the SAN blood supply are the SAN branch, the right intermediate or right posterior atrial branches, and at the left coronary variant--the anterior left, the posterior left and the intermediate left atrial branches. At the even variant the SAN blood supply sources are the right intermediate and the anterior left atrial or the right posterior and the left posterior atrial branches. The data obtained can be used for comparison with the results of coronography to make a skilled analysis of clinical-roentgenological observations.     

Effect of potential fluctuations on the activity of sinoatrial node cells

The effect of fluctuations of the transmembrane potential on the generation of the action potential is studied by simulating the rabbit sinoatrial node (SAN) pacemaker. It is shown that the effect of fluctuations is enhanced with an increase in the concentration of acetylcholine and becomes most pronounced at the border of spontaneous activity loss and after it. When applying and washing off acetylcholine, the hysteretic effect is observed.