Computer three-dimensional reconstruction of the sinoatrial node.

BACKGROUND: There is an effort to build an anatomically and biophysically detailed virtual heart, and, although there are models for the atria and ventricles, there is no model for the sinoatrial node (SAN). For the SAN to show pacemaking and drive atrial muscle, theoretically, there should be a gradient in electrical coupling from the center to the periphery of the SAN and an interdigitation of SAN and atrial cells at the periphery. Any model should include such features. METHODS AND RESULTS: Staining of rabbit SAN preparations for histology, middle neurofilament, atrial natriuretic peptide, and connexin (Cx) 43 revealed multiple cell types within and around the SAN (SAN and atrial cells, fibroblasts, and adipocytes). In contrast to atrial cells, all SAN cells expressed middle neurofilament (but not atrial natriuretic peptide) mRNA and protein. However, 2 distinct SAN cell types were observed: cells in the center (leading pacemaker site) were small, were organized in a mesh, and did not express Cx43. In contrast, cells in the periphery (exit pathway from the SAN) were large, were arranged predominantly in parallel, often expressed Cx43, and were mixed with atrial cells. An approximately 2.5-million-element array model of the SAN and surrounding atrium, incorporating all cell types, was constructed. CONCLUSIONS: For the first time, a 3D anatomically detailed mathematical model of the SAN has been constructed, and this shows the presence of a specialized interface between the SAN and atrial muscle.

Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity.

Recent work on isolated sinoatrial node cells from rabbit has suggested that sarcoplasmic reticulum Ca2+ release plays a dominant role in the pacemaker potential, and ryanodine at a high concentration (30 micromol/L blocks sarcoplasmic reticulum Ca2+ release) abolishes pacemaking and at a lower concentration abolishes the chronotropic effect of beta-adrenergic stimulation. The aim of the present study was to test this hypothesis in the intact sinoatrial node of the rabbit. Spontaneous activity and the pattern of activation were recorded using a grid of 120 pairs of extracellular electrodes. Ryanodine 30 micromol/L did not abolish spontaneous activity or shift the position of the leading pacemaker site, although it slowed the spontaneous rate by 18.9+/-2.5% (n=6). After ryanodine treatment, beta-adrenergic stimulation still resulted in a substantial chronotropic effect (0.3 micromol/L isoproterenol increased spontaneous rate by 52.6+/-10.5%, n=5). In isolated sinoatrial node cells from rabbit, 30 micromol/L ryanodine slowed spontaneous rate by 21.5+/-2.6% (n=13). It is concluded that sarcoplasmic reticulum Ca2+ release does not play a dominating role in pacemaking in the sinoatrial node. The full text of this article is available at http://www.circresaha.org.

Na/K pump alpha subunit expression in rabbit ventricle and regional variations of intracellular sodium regulation.

We examined the isoform distribution and expression of the alpha subunit of the Na/K-ATPase in the left ventricular muscle of rabbit heart in order to determine whether previously reported regional differences in intracellular sodium regulation derive from differences in pump expression. Immunohistochemical techniques show that only the alpha1 isoform is present in rabbit ventricle; therefore, regional variation in isoform distribution is not possible. Western blots of samples taken from subendocardial and sub-epicardial regions confirm the absence of alpha2 and alpha3 isoforms but also show that levels of the alpha1 isoform do not differ significantly in the two regions. The ratio of densitometric readings from blot bands was 1.18+/-0.17 (epicardial:endocardial; mean +/-SEM). Measurements of fully activated pump current in voltage-clamped cells were achieved by dialysing the cell via the patch pipette with 50 mM Na and applying 1 mM dihydroouabain. The current measured was 0.16+/-0.02 pA/pF in epicardial cells and 0.17+/-0.02 pA/pF in endocardial cells. These results indicate that the capacity of cells from the two regions to generate sodium efflux is identical. Regional differences in intracellular sodium regulation, therefore, are more likely to arise from differences in influx of sodium.

Cs+ block of the cardiac muscarinic K+ channel, GIRK1/GIRK4, is not dependent on the aspartate residue at position 173.

Cs+ block of GIRK1/GIRK4 expressed in Xenopus oocytes has been investigated. It has been reported that a negatively charged aspartate residue at position 172 in IRK1 is responsible for Cs+ block of the channel. IRK1, a homotetramer, has four aspartate residues at this position. GIRK1/GIRK4 is a heterotetramer and has two aspartate residues at the equivalent position (GIRK1-D173) and, consequently, it should be less sensitive to Cs+. Cs+ caused voltage-dependent block of GIRK1/GIRK4 current (measured with the two-microelectrode voltage-clamp technique). The apparent fraction of the electrical field through which Cs+ moves in order to reach its site of block (delta approximately equals 1.66) is comparable to that in IRK1, suggesting that Cs+ binds to a similar site in the two channels. GIRK1/GIRK4 was less sensitive than IRK1 to Cs+ -the Kd was 3.0-8.5 times greater and at potentials more negative than approximately or = to 130 mV there was voltage-dependent relief of block of GIRK1/GIRK4 (not the case with IRK1). However, the mutations GIRK1-D173A and GIRK1-D173Q increased the sensitivity of the channel to Cs+, while adding a negatively charged aspartate residue to GIRK4 at the equivalent position (GIRK4-N 79D) decreased Cs+ sensitivity. GIRK1-D173 cannot be the site of Cs+ block of GIRK1/GIRK4.

Residues and mechanisms for slow activation and Ba2+ block of the cardiac muscarinic K+ channel, Kir3.1/Kir3.4.

Mechanisms and residues responsible for slow activation and Ba(2+) block of the cardiac muscarinic K(+) channel, Kir3.1/Kir3.4, were investigated using site-directed mutagenesis. Mutagenesis of negatively charged residues located throughout the pore of the channel (in H5, M2, and proximal C terminus) reduced or abolished slow activation. The strongest effects resulted from mutagenesis of residues in H5 close to the selectivity filter; mutagenesis of residues in M2 and proximal C terminus equivalent to those identified as important determinants of the activation kinetics of Kir2.1 was less effective. In giant patches, slow activation was present in cell-attached patches, lost on excision of the patch, and restored on perfusion with polyamine. Mutagenesis of residues in H5 and M2 close to the selectivity filter also decreased Ba(2+) block of the channel. A critical residue for Ba(2+) block was identified in Kir3.4. Mutagenesis of the equivalent residue in Kir3.1 failed to have as pronounced an effect on Ba(2+) block, suggesting an asymmetry of the channel pore. It is concluded that slow activation is principally the result of unbinding of polyamines from negatively charged residues close to the selectivity filter of the channel and not an intrinsic gating mechanism. Ba(2+) block involves an interaction with the same residues.

Changes in contraction, cytosolic Ca2+ and pH during metabolic inhibition and upon restoration of mitochondrial respiration in rat ventricular myocytes.

Exposure of cardiac muscle to metabolic poisons reduces the availability of cellular ATP and cardiac dysfunction ensues. In this study rat ventricular myocytes were exposed to 2-deoxyglucose, iodoacetate and cyanide to induce complete metabolic blockade. Changes in contraction, cytosolic Ca2+ and pH were determined during metabolic blockade and following restoration of mitochondrial ATP production. Metabolic blockade resulted in a rapid failure of contractions and Ca2+ transients, a rise of diastolic Ca2+, a cytosolic acidosis and ultimately a rigor contracture. Washing out cyanide during the development of the rigor contracture led to a rapid relaxation of the contracture, a fall in cytosolic Ca2+ and a rapid, partial reversal of the cytosolic acidosis. The partial reversal of the cytosolic acidosis and fall of cytosolic Ca2+ were abolished in the presence of oligomycin. This suggests that the rapid partial recovery of cytosolic acidosis could result from the rephosphorylation of ADP to ATP by the mitochondrial F1,F0-ATPase (a reaction that consumes protons).

Regional differences in the regulation of intracellular sodium and in action potential configuration in rabbit left ventricle.

In this study we report measurements of the intracellular sodium concentration ([Na+]i) in cardiac myocytes isolated from sub-endocardial and sub-epicardial regions of the rabbit left ventricle. These measurements show that [Na+]i is significantly higher in sub-epicardial than in sub-endocardial myocytes both at rest and during steady-state stimulation at 0.5 Hz. During a 10-min rest the rate of fall of [Na+]i was identical in cells from the two regions but during post-rest recovery [Na+]i rose significantly faster in sub-endocardial cells. The effect that this difference in sodium regulation may have on the rate of recovery of the calcium transient post-rest is discussed. The steady-state differences in [Na+]i do not appear to have the effect on contraction we would expect i.e. steady-state contraction is larger in sub-endocardial cells where [Na+]i is lower. Changes in [Na+]i brought about by altering the frequency of stimulation are associated with the expected changes of contraction. Action potentials were found to be significantly longer in sub-endocardial cells and following rest action potential duration was shortened although regional differences were maintained. No regional differences in this effect of rest or in recovery of the action potential post-rest were found.

The effects of levosimendan on [Ca2+]i in guinea-pig isolated ventricular myocytes.

Ca2+-sensitising agents offer a new approach to the treatment of congestive heart failure. This study examined the effects of the Ca2+-sensitising agent, levosimendan, on contraction and [Ca2+]i in guinea-pig ventricular myocytes. Levosimendan 100 nM produced an increase in cell shortening without affecting the [Ca2+]i transient or the Ca2+ content of the sarcoplasmic reticulum. 1 microM levosimendan increased the rate of decay of the [Ca2+]i transient and increased the Ca2+ content of the sarcoplasmic reticulum. These results suggest that at therapeutically relevant concentrations levosimendan can produce a significant inotropic effect without affecting [Ca2+]i but at higher concentrations may also inhibit phosphodiesterase.

Variability of spontaneous Ca2+ release between different rat ventricular myocytes is correlated with Na(+)-Ca2+ exchange and [Na+]i.

We have studied the factors responsible for the variation of the frequency of "waves" caused by spontaneous Ca2+ release in rat ventricular myocytes. The experiments were performed in isolated myocytes using the fluorescent indicators Indo-1 (to measure [Ca2+]i) and SBFI (to measure [Na+]i). After electrical stimulation (either with action potentials or voltage-clamp pulses), some cells showed spontaneous Ca2+ release. The frequency of this release, where present, was variable. The Ca2+ content of the sarcoplasmic reticulum (SR) was measured by applying caffeine (10 mmol/L). The resulting increase of [Ca2+]i activated the electrogenic Na(+)-Ca2+ exchange, and the integral of this current was used to estimate the Ca2+ content of the SR. The SR Ca2+ content was significantly higher in cells that oscillated at high rates ( > 10 . min-1) than in those that were quiescent. The rate of removal of Ca2+ from the cytoplasm by non-SR mechanisms was measured by adding caffeine (10 mmol/L) and measuring the rate constant of decay of the resulting increase of [Ca2+]i. Cells that had a high rate constant of decay of [Ca2+]i had a low frequency of oscillations. Measurements of [Na+]i showed a positive correlation between the frequency of spontaneous SR Ca2+ release and [Na+]i. After cessation of stimulation, there was a gradual decrease of [Na+]i, which was correlated with a parallel decrease of the frequency of oscillation rate. We conclude that the variability of frequency of spontaneous SR Ca2+ release is due to variations of the rate of Ca2+ removal from the cell, which are probably due to Na(+)-Ca2+ exchange. The variability of Na(+)- Ca2+ exchange rate, in turn, is likely to result from variations of [Na+]i.