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.     

Selected contribution: axial stretch increases spontaneous pacemaker activity in rabbit isolated sinoatrial node cells.

Isolated, spontaneously beating rabbit sinoatrial node cells were subjected to longitudinal stretch, using carbon fibers attached to both ends of the cell. Their electrical behavior was studied simultaneously in current-clamp or voltage-clamp mode using the perforated patch configuration. Moderate stretch ( approximately 7%) caused an increase in spontaneous beating rate (by approximately 5%) and a reduction in maximum diastolic and systolic potentials (by approximately 2.5%), as seen in multicellular preparations. Mathematical modeling of the stretch intervention showed the experimental results to be compatible with stretch activation of cation nonselective ion channels, similar to those found in other cardiac cell populations. Voltage-clamp experiments validated the presence of a stretch-induced current component with a reversal potential near -11 mV. These data confirm, for the first time, that the positive chronotropic response of the heart to stretch is, at least in part, encoded on the level of individual sinoatrial node pacemaker cells; all reported data are in agreement with a major contribution of stretch-activated cation nonselective channels to this response.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

Effect of hydrogen peroxide on the membrane currents of sinoatrial node cells from rabbit heart

The effects of H(2)O(2) on pacemaker activity and underlying membrane currents were studied in isolated rabbit sinoatrial (SA) node cells using perforated patch current- and voltage-clamp methods. Short-term exposure (<10 min) of the nodal cells to H(2)O(2) (200 microM) resulted in an initial shortening of spontaneous action potential cycle length (from 445 +/- 60 to 398 +/- 56 ms; P < 0.05) and a prolongation of action potential duration. H(2)O(2) (100 microM) significantly increased peak L-type Ca(2+) current (I(Ca,L)) from -384 +/- 77 to -439 +/- 84 pA (116 +/- 2%, n = 6). Additionally, the persistent or non-inactivating component of I(Ca,L) was increased from -52 +/- 3 to -88 +/- 14 pA (174 +/- 19%, n = 6). The hyperpolarization-activated current (I(f)) was decreased from -228 +/- 62 to -161 +/- 72 pA after exposure to H(2)O(2) (n = 7). There were no changes in the delayed rectifier K(+) current (I(K)) (n = 7). H(2)O(2)-induced Ca(2+) currents were blocked by 2 microM nicardipine (n = 6), 2 mM Ni(2+) (n = 2), and the protein kinase C (PKC) inhibitor bisindolylmaleimide (10(-7) M; n = 4) but not by 20 microM tetrodotoxin. These results suggest that H(2)O(2) can increase the spontaneous pacing rate in rabbit SA node cells by enhancing I(Ca,L) and that this effect is mediated by a PKC-dependent pathway.     

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.     

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.     

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.     

Minor contribution of cytosolic Ca2+ transients to the pacemaker rhythm in guinea pig sinoatrial node cells

The question of the extent to which cytosolic Ca(2+) affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the "Ca(2+) clock" (C) and "membrane clock" (M) hypotheses, together with patch-clamp experiments in isolated guinea pig sinoatrial node cells. By applying lead potential analysis to the models, the C mechanism, which is dependent on potentiation of Na(+)/Ca(2+) exchange current via spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) during diastole, was found to overlap M mechanisms in the C model. Rapid suppression of pacemaker rhythm was observed in the C model by chelating intracellular Ca(2+), whereas the M model was unaffected. Experimental rupturing of the perforated-patch membrane to allow rapid equilibration of the cytosol with 10 mM BAPTA pipette solution, however, failed to decrease the rate of spontaneous action potential within ～30 s, whereas contraction ceased within ～3 s. The spontaneous rhythm also remained intact within a few minutes when SR Ca(2+) dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca(2+) dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca(2+)-mediated inactivation of the L-type Ca(2+) currents induced by BAPTA, were well explained by assuming Ca(2+) chelation, even in the proximity of the channel pore in addition to the bulk cytosol in the M model. Taken together, the experimental and model findings strongly suggest that the C mechanism explicitly described by the C model can hardly be applied to guinea pig sinoatrial node cells. The possible involvement of L-type Ca(2+) current rundown induced secondarily through inhibition of Ca(2+)/calmodulin kinase II and/or Ca(2+)-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca(2+) concentration reduction for >5 min.     

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.