Lennart N. Bouman

Functional and morphological organization of the rabbit sinus node.

In isolated right atria of the rabbit heart, we studied the activation pattern within the sinus node, using the microelectrode technique. After the electrophysiological experiments, the prep-arations were subjected to a correlative morphological investigation, using light or electron microscopy. Different criteria for defining the dominant pacemaker were compared. A group of at least 5000 cells, located within the central part of the node where the most characteristic tissue architecture was found, was considered to be responsible for generation of the impulse. At the ultrastructural level, this leading cell group appeared to be part of a larger uniform cell group. The number of gap junctions observed suggests that all nodal cells are coupled by these structures. Toward the periphery, the excitation wave was propagated preferentially in an oblique cranial direction toward the crista terminalis. Neither morphologically nor electrophysiologically specific pathways were found for the conduction, but the preferential direction could be explained by the tissue architecture. Circ Res: 46:11-22, 1980

Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution

OBJECTIVE The sinoatrial (SA) node consists of a relatively small number of poorly coupled cells. It is not well understood how these pacemaker cells drive the surrounding atrium and at the same time are protected from its hyperpolarizing influence. To explore this issue on a small tissue scale we studied the activation pattern of the mouse SA node region and correlated this pattern with the distribution of different gap junction proteins, connexin (Cx)37, Cx40, Cx43 and Cx45. METHODS AND RESULTS The mouse SA node was electrophysiologically mapped using a conventional microelectrode technique. The primary pacemaker area was located in the corner between the lateral and medial limb of the crista terminalis. Unifocal pacemaking occurred in a group of pacemaking fibers consisting of 450 cells. In the nodal area transitions of nodal and atrial waveform were observed over small distances ( approximately 100 microm). Correlation between the activation pattern and connexin distribution revealed extensive labeling by anti-Cx45 in the primary and secondary pacemaker area. Within these nodal areas no gradient in Cx45 labeling was found. A sharp transition was found between Cx40- and Cx43-expressing myocytes of the crista terminalis and the Cx45-expressing myocytes of the node. In addition, strands of myocytes labeled for Cx43 and Cx40 protrude into the nodal area. Cx37 labeling was only present between endothelial cells. Furthermore, a band of connective tissue largely separates the nodal from the atrial tissue. CONCLUSIONS Our results demonstrate strands of Cx43 and Cx40 positive atrial cells protruding into the Cx45 positive nodal area and a band of connective tissue largely separating the nodal and atrial tissue. This organization of the mouse SA node provides a structural substrate that both shields the nodal area from the hyperpolarizing influence of the atrium and allows fast action potential conduction from the nodal area into the surrounding atrium.

Effects of Delayed Rectifier Current Blockade by E-4031 on Impulse Generation in Single Sinoatrial Nodal Myocytes of the Rabbit

The role of the delayed rectifier current (IK) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit. We used the class III antiarrhythmic drug E-4031, which blocks IK in rabbit ventricular myocytes. In single sinoatrial nodal cells, E-4031 (0.1 mumol/L) significantly prolonged cycle length and action potential duration, depolarized maximum diastolic potential, and reduced both the upstroke velocity of the action potential and the diastolic depolarization rate. Half of the cells were arrested completely. At higher concentrations (1 and 10 mumol/L), spontaneous activity ceased in all cells. Three ionic currents fundamental for pacemaking, ie, IK, the long-lasting inward calcium current (ICa,L), and the hyperpolarization-activated current (I(f)), were studied by using the whole-cell and amphotericin-perforated patch technique. E-4031 blocked part of the outward current during depolarizing steps as well as the tail current upon subsequent repolarization (ITD) in a dose-dependent manner. E-4031 (10 mumol/L) depressed ITD (88 +/- 4%) (n = 6), reduced peak ICa,L at 0 mV (29 +/- 15%) (n = 4), but did not affect I(f). Lower concentrations did not affect ICa,L. Additional use of 5 mumol/L nifedipine demonstrated that ITD is carried in part by a calcium-sensitive current. Interestingly, complete blockade of IK and ICa,L unmasked the presence of a background current component with a reversal potential of -32 +/- 5.4 mV (n = 8) and a conductance of 39.5 +/- 5.6 pS/pF, which therefore can contribute both to the initial part of repolarization and to full diastolic depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of L-type Ca2+current to electrical activity in sinoatrial nodal myocytes of rabbits

The role of L-type calcium current ( I Ca,L) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit, with the use of the amphotericin-perforated patch-clamp technique. Nifedipine, at a concentration of 5 μM, was used to block I Ca,L. At this concentration, nifedipine selectively blocked I Ca,L for 81% without affecting the T-type calcium current ( I Ca,T), the fast sodium current, the delayed rectifier current ( I K), and the hyperpolarization-activated inward current. Furthermore, we did not observe the sustained inward current. The selective action of nifedipine on I Ca,L enabled us to determine the activation threshold of I Ca,L, which was around -60 mV. As nifedipine (5 μM) abolished spontaneous activity, we used a combined voltage- and current-clamp protocol to study the effects of I Ca,L blockade on repolarization and diastolic depolarization. This protocol mimics the action potential such that the repolarization and subsequent diastolic depolarization are studied in current-clamp conditions. Nifedipine significantly decreased action potential duration at 50% repolarization and reduced diastolic depolarization rate over the entire diastole. Evidence was found that recovery from inactivation of I Ca,L occurs during repolarization, which makes I Ca,L available already early in diastole. We conclude that I Ca,L contributes significantly to the net inward current during diastole and can modulate the entire diastolic depolarization.

Effect of an Early Atrial Premature Beat on Activity of the Sinoatrial Node and Atrial Rhythm in the Rabbit

In the spontaneously beating isolated right atrium of the rabbit, premature beats were elicited by electrical stimulation. When the premature beat was elicited early in the atrial cycle, the postextrasystolic pause had a short duration and the sum of the pre- and postextrasystolic pause was about the same as the duration of a normal spontaneous interval. We have tried to demonstrate, using simultaneous multiple microelectrode impalements of SA node fibers, that by such an early atrial premature beat the atrium could be activated by a reentrant mechanism. The results of our experiments led us to the conclusion that the impulse of the premature beat, elicited early in the atrial cycle, discharges the SA node only fractionally and that the fibers in the neighborhood of the activated area are influenced electrotonically. This causes a change of both the site and the moment of the spontaneous impulse formation. The SA node discharges spontaneously after such an early premature beat. Reentry activation is likely to occur when a series of atrial premature beats is observed. A supraventricular tachvcardia might be caused by repeated atrial discharges following a stimulus very shortly after the atrial refractory period.

Delayed Rectifier Channels in Human Ventricular Myocytes

BACKGROUND Previous studies have shown that in heart there are two kinetically distinct components of delayed rectifier current: a rapidly activating component (IKr) and a more slowly activating component (IKs). The presence of IKr and/or IKs appears to be species dependent. We studied the nature of the delayed rectifier current in human ventricle in whole-cell and single-channel experiments. METHODS AND RESULTS Ventricular myocytes were obtained from hearts of patients with ischemic or dilated cardiomyopathy. Single-channel currents and whole-cell tail currents were recorded at negative potentials directly after return from a depolarizing step. Single-channel currents were measured in the cell-attached patch configuration with 140 mmol/L K+ in the pipette. In the present study, we identified a voltage-dependent channel with a single-channel conductance of 12.9 +/- 0.8 pS (mean +/- SEM, n = 5) and a reversal potential near to the K+ equilibrium potential, suggesting that the channel is selective to K+ ions. Channel activity was observed only after a depolarizing step and increased with the duration and amplitude of the depolarization, indicating time- and voltage-dependent activation. Activation at +30 mV was complete within 300 milliseconds, and the time constant of activation, determined in the whole-cell configuration, was 101 +/- 25 milliseconds (mean +/- SEM, n = 4). The voltage dependence of activation could be described by a Boltzmann equation with a half-activation potential of -29.9 mV and a slope factor of 9.5 mV. The addition of the class III antiarrhythmic drug E-4031 completely blocked channel activity in one patch. No indications for the presence of IKs were found in these experiments. CONCLUSIONS The conformity between the properties of IKr and those of the K+ channel in the present study strongly suggests that IKr is present in human ventricle.

Spatial and functional relationship between myocytes and fibroblasts in the rabbit sinoatrial node

In an attempt to understand better the directional differences in conduction velocity in the rabbit sinoatrial node, a possible conductive role of the abundant connective tissue surrounding the myocytes has been investigated. In particular, starting from the finding of communicating junctions between heart muscle cells and fibroblasts in tissue culture, heterologous gap junctions were searched for in thin sections of the rabbit sinoatrial node. Within and at the edge of nodal cell clusters, fibroblasts often show thin sheet-like extensions parallel to the surface of myocytes. In contrast to the intimately contacting myocytes, fibroblast extensions are kept separated from the myocytes by the basement membrane of the latter. Besides some rare undefined membrane appositions a single tiny gap junction-like structure was found between a fibroblast and a myocyte in a tissue area in which the calculated number of gap junctions between myocytes amounts from 1.10(4) to 3.10(4). Yet, fibroblasts are linked together regularly by small gap junctions containing a wider gap than the junctions between the myocytes (1.4 +/- 0.4 nm vs. 1.0 +/- 0.4 nm, resp., P less than 0.05). As an alternative to direct electrical coupling, the possibility of interaction between fibroblasts and nodal cells by capacitive coupling has been considered. Model calculations based on the reconstruction of some fibroblast extensions parallel to nodal cells show that the current which can be transmitted from discharging nodal cells to fibroblasts is negligible. It is concluded that fibroblasts do not participate in the impulse conduction within the sinoatrial node. The origin of the directional differences in conduction velocity in the sinoatrial node must be found in the spatial arrangement of the myocytes and the distribution of the gap junctions between these cells only.

Pacemaker cell types in the rabbit sinus node: A correlative ultrastructural and electrophysiological study

In isolated preparations of the rabbit sinus node, we have investigated the fine structure of the tissue at many sites which had been electro-physiologically identified by means of microelectrode recordings. For each of these sites we quantified the myofilament density of the cells, since this appeared to be a useful parameter for characterizing cell types in the sinus node region; and because myofilaments were present both in isolated form and organized in myofibrils, the degree of organization was also measured in a semi-quantitative fashion. The electrical activity of cells at a given site was characterized by the activation moment relative to the cardiac cycle and furthermore by the rate of diastolic depolarization and the maximum rate of rise of the action potential. From the centre of the node toward the periphery a very gradual increase in myofilament density was observed in all directions. It was found that the rate of diastolic depolarization, which feature is generally accepted as being basic to the automaticity of the sinus node, was inversely related to the volume percentage of myofilaments. This means that a relation exists between the pacemaker action and the cell type. The anatomically less developed cells, i.e. the cells with the lowest density of organelles, which are located in the central portion of the node, are the most specialized pacemakers. No clear relation was found between the myofilament density and the rate of rise of the action potential. In the direction of the crista terminalis we observed an increase in the rate of rise and an increase in conduction velocity concomitant with the increase in myofilament density. Toward the interatrial septum, however, the increase in myofilament density was not accompanied by an increase in rate of rise; in this direction the impulse conduction was blocked. A correlation between cell type and impulse conduction could thus not be established.

Single delayed rectifier channels in the membrane of rabbit ventricular myocytes.

In rabbit ventricular cells, the delayed rectifier current (IK) has not been extensively studied, and properties of single IK channels still need to be determined. In this study, we present data on a voltage-dependent channel in rabbit ventricular cells; the properties indicate that it is an IK channel. Patch-clamp experiments were carried out on cell-attached and inside-out patches of rabbit ventricular cells. Single-channel currents were recorded at negative potentials as inward currents with 150 mM K+ in the pipette. Voltage-dependent channel activity was only present after the return from a depolarizing test pulse, indicating activation on depolarization. Single-channel conductance calculated from the current-voltage relation was 13.1 pS (pooled data, n = 8). The shift in reversal potential of the unitary currents, determined at 150 and 300 mM K+ at the intracellular side of the membrane, showed that the channels were highly permeable to potassium ions. Increase of the duration or the amplitude of the depolarizing test pulse increased channel activity. The time constant for activation at +30 mV was 187 msec (pooled data, n = 4). Half-activation potential was -4.9 +/- 3.8 mV (mean +/- SD), and the slope factor was 7.2 +/- 3.7 mV (mean +/- SD). Current tails, reconstructed from averaged single-channel currents, revealed that the time course of deactivation decreased from 694 +/- 73 msec at -80 mV to 136 +/- 39 msec at -110 mV. Additional evidence that the channel was indeed an IK channel was provided by the observation that the channel was blocked by 10(-7) M E-4031, a class III antiarrhythmic agent that has been shown to block a component of the macroscopic IK in guinea pig heart.

Sinus node and atrium cells from the rabbit heart: A quantitative electron microscopic description after electrophysiological localization

Abstract Electrophysiologically identified cell groups in the sinus node from the rabbit have been compared with atrial fibers with the electron microscope. The point counting method has been used to estimate the volume density of the following structures: nucleus, mitochondria, myofilaments, sarcoplasmic reticulum tubules and subsarcolemmal vesicles. These data were collected in leading pacemaker cells, latent pacemaker cells and atrium cells from the crista terminalis. It has been found that organized structures in leading pacemaker cells occupy about 50% of the cell volume, as compared with over 90% in atrial fibers. Leading pacemaker cells consequently appear extremely “empty”. It has also been found that the group of cells which show the characteristic features of leading pacemaker cells at the ultrastructural level as observed in the correlated experiments is larger than the leading center found in electrophysiology and thus it seems impossible with the actual observation methods to delineate the leading pacemaker center using only cytological criteria.