Sinoatrial Node Pacemaker Cells Research Articles

Pacemaker activity and ionic currents in mouse atrioventricular node cells

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of INa by tetrodotoxin (TTX) or of ICa,L by isradipine abolished AVNCs pacemaker activity. TTX-resistant (INar) and TTX-sensitive (INas) Na+ currents were recorded in mouse AVNCs, as well as T- (ICa,T) and L-type (ICa,L) Ca2+ currents ICa,L density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (If) and that of the fast component of the delayed rectifier current (IKr) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of If by 3 µM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for If in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K+ current (Ito). In contrast, no detectable slow component of the delayed rectifier current (IKs) could be recorded in AVNCs. The lower densities of If and ICa,L, as well as higher expression of IKr in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.

Angiotensin II decreases spontaneous firing rate of guinea-pig sino-atrial node cells

Angiotensin II (Ang II) plays an important role in the regulation of cardiac function, but its electrophysiological effects on sino-atrial (SA) node are not well understood. In this study, the immediate effect of Ang II on action potentials and ionic currents were investigated by using whole-cell patch-clamps in single guinea-pig SA node pacemaker cells. We demonstrated that Ang II exerted a negative effect on spontaneous firing rate, with a concomitant reduction in the slope of diastolic depolarization. The inhibitory effect of Ang II on spontaneous activity displayed a concentration-dependent manner in the range of 1–1000nM, with IC50 of 8.34nM. Ang II type 1 (AT1) receptor antagonist valsartan (1μM) abolished the inhibitory effect. In contrast, Ang II type 2 (AT2) receptor antagonist, PD123319 (1μM) didn't affect the action of Ang II. Ang II had no significant effect on hyperpolarization-activated current (If) in SA node cells. However, it significantly slowed the deactivation of the slowly activated delayed rectifier K+ current (Iks) and increased the tail current density. Furthermore, Ang II decreased the current density of L-type Ca2+ current in SA node cells. Our data demonstrate that Ang II reduces the auto rhythm of SA node cells via enhancing Iks and reducing ICaL. The result suggests a potential mechanism by which elevated levels of Ang II may be involved in the occurrence of SA node dysfunction in cardiac pathophysiology.

Implantation of Sinoatrial Node Cells Into Canine Right Ventricle: Biological Pacing Appears Limited by the Substrate

Biological pacing has been proposed as a physiologic counterpart to electronic pacing, and the sinoatrial node (SAN) is the general standard for biological pacemakers. We tested the expression of SAN pacemaker cell activity when implanted autologously in the right ventricle (RV). We induced complete heart block and implanted electronic pacemakers in the RV of adult mongrel dogs. Autologous SAN cells isolated enzymatically were studied by patch clamp to confirm SAN identity. SAN cells (400,000) were injected into the RV subepicardial free wall and dogs were monitored for 2 weeks. Pacemaker function was assessed by overdrive pacing and IV epinephrine challenge. SAN cells expressed a time-dependent inward current (I(f)) activating on hyperpolarization: density = 4.3 ± 0.6 pA/pF at -105 mV. Four of the six dogs demonstrated >50% of beats originating from the implant site at 24 h. Biological pacemaker rates on days 7-14 = 45-55 bpm and post-overdrive escape times = 1.5-2.5 s. Brisk catecholamine responsiveness occurred. Dogs implanted with autologous SAN cells manifest biological pacing properties dissimilar from those of the anatomic SAN. This highlights the importance of cell and substrate interaction in generating biological pacemaker function.

Cav1.3 L-Type Calcium Channels-Mediated Ryanodine Receptor Dependent Calcium Release Controls Heart Rate

Pacemaker activity of the sino-atrial node (SAN) controls heart rate. However, the mechanism underling SAN pacemaker activity is not completely understood and in particular, the respective physiological importance of ion channels and ryanodine receptor (RyR)-dependent Ca2+ release in pacemaking is hotly debated. We have investigated Ca2+ handling in SAN pacemaker cells of wild-type (WT) and mice lacking L-type Cav1.3 (Cav1.3-/-) channels. In isolated Cav1.3-/- SAN cells the frequency of Ca2+ transients was reduced by 45% compared to WT pacemaker cells. Loss of Cav1.3 channels also blunted by about 47% the positive chronotropic effect induced by 0.01 μM isoproterenol (ISO). Furthermore, in Cav1.3-/- pacemaker cells, local Ca2+ release (LCR) occurring during the diastolic phase was reduced by 71%. In SAN cells from mice in which L-type Cav1.2 channels have been rendered insensitive to dihydropyridines (Cav1.2DHP-/-), application of 0.3 μM isradipine decreased diastolic LCR by 78 %, thus showing that Cav1.3 channels are major regulators of RyR-dependent LCR during the diastolic phase. In individual cells of isolated intact SAN, pacemaking of Cav1.3-/- cells was characterized by reduced (37%) frequency of Ca2+ transients and an increase in Ca2+ waves. Normal pacemaking in Cav1.3-/- isolated SAN cells and intact tissue could be observed only after direct activation of RyR-dependent Ca2+ release by low doses of caffeine (200 μM). Experiments with high doses of caffeine (10 mM) in Cav1.3-/- cells, showed that the reduction in diastolic LCR and in the frequency Ca2+ transients could not be ascribed to a decrease in sarcoplasmic reticulum (SR) Ca2+ content. Our results show that in SAN pacemaker cell, LCR Ca2+ release is tightly controlled by Cav1.3 channels and that such a control is critical for promoting the formation of whole-cell Ca2+ transients.Support: FWF (P20670, P22528), ANR-06-PHYSIO-004-01.

If and SR Ca2+ release both contribute to pacemaker activity in canine sinoatrial node cells

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a ‘voltage clock’ and a Ca2+ dependent process, or ‘Ca2+ clock.’ The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (If) is thought to be particularly important. A Ca2+ dependent process triggers APs when sarcoplasmic reticulum (SR) Ca2+ release activates inward current carried by the forward mode of the electrogenic Na+/Ca2+ exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca2+ clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective If antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest (∼14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca2+ release, but did not affect basal or isoproterenol-enhanced If. Taken together, these results indicate that voltage and Ca2+ dependent automaticity mechanisms coexist in canine SAN cells, and suggest that If and SR Ca2+ release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.

A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart's pacemaker.

Ion channels on the surface membrane of sinoatrial nodal pacemaker cells (SANCs) are the proximal cause of an action potential. Each individual channel type has been thoroughly characterized under voltage clamp, and the ensemble of the ion channel currents reconstructed in silico generates rhythmic action potentials. Thus, this ensemble can be envisioned as a surface "membrane clock" (M clock). Localized subsarcolemmal Ca(2+) releases are generated by the sarcoplasmic reticulum via ryanodine receptors during late diastolic depolarization and are referred to as an intracellular "Ca(2+) clock," because their spontaneous occurrence is periodic during voltage clamp or in detergent-permeabilized SANCs, and in silico as well. In spontaneously firing SANCs, the M and Ca(2+) clocks do not operate in isolation but work together via numerous interactions modulated by membrane voltage, subsarcolemmal Ca(2+), and protein kinase A and CaMKII-dependent protein phosphorylation. Through these interactions, the 2 subsystem clocks become mutually entrained to form a robust, stable, coupled-clock system that drives normal cardiac pacemaker cell automaticity. G protein-coupled receptors signaling creates pacemaker flexibility, ie, effects changes in the rhythmic action potential firing rate, by impacting on these very same factors that regulate robust basal coupled-clock system function. This review examines evidence that forms the basis of this coupled-clock system concept in cardiac SANCs.

Synchronization of Spontaneous Stochastic RyR Activation in Ventricular Myocytes (VM) by Camp or Disengagement of Phospholambam (PLB) From SERCA2

Stochastic RyR activation underlies Ca2+ sparks in VM. Here we show that in saponin “skinned” VM, bathed in 100 nM Ca2+ at 35°C, cAMP converts this stochastic spontaneous RyR activation (Ca2+ sparks-confocal linescan imaging) into synchronized, rhythmic RyR activation (Fig. A) about an average dominant frequency 2.2 ± 0.13 Hz (n=3). Of note, cAMP does not alter the SR Ca2+ load assessed by the rapid application of caffeine (107 ± 17.3 nM Ca2+ n=9 prior and 121 ± 19.3 nM Ca2+ n=3 during cAMP exposure). When Ca2+ pumping into SR is selectively accelerated by a PLB antibody (2D12, 0.013 mg/ml) that disengages PLB from SERCA2, stochastic RyR Ca2+ release becomes rhythmic (Fig. B) about an average dominant frequency of 2.6 + 0.21 Hz (n=5). The amplitude of the integrated spontaneous Ca2+ release signal during any given epoch increases when stochastic RyR activation becomes synchronized, i.e. converted to rhythmic activation (Fig. C). This cAMP-generated rhythmicity of spontaneous RyR activation of VM mimics rhythmic spontaneous diastolic Ca2+ releases in sinoatrial nodal pacemaker cells which have a basal high level of intrinsic cAMP-dependent signaling.View Large Image | View Hi-Res Image | Download PowerPoint Slide

A Novel Method to Quantify Contribution of Channels and Transporters to Membrane Potential Dynamics

The action potential, once triggered in ventricular or atrial myocytes, automatically proceeds on its time course or is generated spontaneously in sinoatrial node pacemaker cells. It is induced by complex interactions among such cellular components as ion channels, transporters, intracellular ion concentrations, and signaling molecules. We have developed what is, to our knowledge, a new method using a mathematical model to quantify the contribution of each cellular component to the automatic time courses of the action potential. In this method, an equilibrium value, which the membrane potential is approaching at a given moment, is calculated along the time course of the membrane potential. The calculation itself is based on the time-varying conductance and the reversal potentials of individual ion channels and electrogenic ion transporters. Since the equilibrium potential moves in advance of the membrane potential change, we refer to it as the lead potential, VL. The contribution of an individual current was successfully quantified by comparing dVL/dt before and after fixing the time-dependent change of a component of interest, such as the variations in the open probability of a channel or the turnover rate of an ion transporter. In addition to the action potential, the lead-potential analysis should also be applicable in all types of membrane excitation in many different kinds of cells.

Main Regularities of Mutual Location of Different Pacemaker Cells in the Rat Heart Sinoatrial Node

The distribution of pacemaker cells and atrial working cardiomyocytes in the anterior right atrial wall was studied using intracellular glass microelectrodes under conditions of short-term culturing. It was shown that the dominant pacemaker region and the functional tail are located in the lateral area of the central part of the sinoatrial node, while the medial area is occupied by latent pacemaker cells.

Ivabradine

Ivabradine

Control of heart rate by cAMP sensitivity of HCN channels

"Pacemaker" f-channels mediating the hyperpolarization-activated nonselective cation current I(f) are directly regulated by cAMP. Accordingly, the activity of f-channels increases when cellular cAMP levels are elevated (e.g., during sympathetic stimulation) and decreases when they are reduced (e.g., during vagal stimulation). Although these biophysical properties seem to make f-channels ideal molecular targets for heart rate regulation by the autonomic nervous system, the exact contribution of the major I(f)-mediating cardiac isoforms HCN2 and HCN4 to sinoatrial node (SAN) function remains highly controversial. To directly investigate the role of cAMP-dependent regulation of hyperpolarization activated cyclic nucleotide activated (HCN) channels in SAN activity, we generated mice with heart-specific and inducible expression of a human HCN4 mutation (573X) that abolishes the cAMP-dependent regulation of HCN channels. We found that hHCN4-573X expression causes elimination of the cAMP sensitivity of I(f) and decreases the maximum firing rates of SAN pacemaker cells. In conscious mice, hHCN4-573X expression leads to a marked reduction in heart rate at rest and during exercise. Despite the complete loss of cAMP sensitivity of I(f), the relative extent of SAN cell frequency and heart rate regulation are preserved. Our data demonstrate that cAMP-mediated regulation of I(f) determines basal and maximal heart rates but does not play an indispensable role in heart rate adaptation during physical activity. Our data also reveal the pathophysiologic mechanism of hHCN4-573X-linked SAN dysfunction in humans.

Glycyrretinic acid blocks cardiac sodium channels expressed in Xenopus oocytes

Ethnopharmacological relevance Licorice has been used to treat many ailments including cardiovascular disorders in China for long time . Recent studies have shown that the cardiac actions of licorice have been attributed to its active component, glycyrretinic acid (GA). However, its mechanism remains poorly understood. Aim of the Study The effects of GA on the cardiac sodium currents ( I Na), L-type calcium currents ( I Ca,L) and hyperpolarization-activated inward currents ( I f) were investigated. Materials and methods Human isoforms of wild-type and ΔKPQ-mutant type sodium channels were expressed in Xenopus oocytes, and the resulting currents (peak and late I Na) were recorded using a two-microelectrode voltage-clamp technique. A perforated patch clamp technique was employed to record I Ca,L and I f from isolated rabbit sinoatrial node pacemaker cells. Results GA inhibited peak I Na (33% at 90 μM) and late I Na (72% at 90 μM), but caused no significant effects on I Ca,L and I f. Conclusion GA blocked cardiac sodium currents, particularly late I Na. Our findings might help to understand the traditional use of licorice in the treatment of cardiovascular disorders, because reduction of sodium currents (particularly late I Na) would be expected to provide protection from Na +-induced Ca 2+ overload and cell damage.

Heart rate control: f-channel blockers

This review is devoted to the problem of pharmacological heart rate (HR) control in patients with cardiovascular diseases. Analysis of the literature allows the conclusion that an increased HR at rest (more than 80 bpm) may be regarded as an independent risk factor for both overall and cardiovascular mortality. Naturally, HR decrease is one of the most important goals of combination therapy for cardiovascular diseases, in particular, coronary heart disease (CHD). One of the possible approaches to the solution of this task is to develop and introduce into clinical practice medications capable of inhibiting the automatism of sinoatrial node pacemaker cells by blocking specialized potential-dependent transmembrane f-channels in the pacemaker cell membrane. After the mechanism of action, this group of agents are named f channel blockers. The electrophysiological mechanisms responsible for spontaneous diastolic depolarization of sinoatrial node pacemaker cells, as well as the problems of the fundamental, clinical, and evidence-based pharmacology of f -blockers (exemplified by Ivabradine), are considered in the review.

Is sodium current present in human sinoatrial node cells?

Pacemaker activity of the sinoatrial node has been studied extensively in various animal species, but is virtually unexplored in man. As such, it is unknown whether the fast sodium current (I(Na)) plays a role in the pacemaker activity of the human sinoatrial node. Recently, we had the unique opportunity to perform patch-clamp experiments on single pacemaker cells isolated from a human sinoatrial node. In 2 out of the 3 cells measured, we observed large inward currents with characteristics of I(Na). Although we were unable to analyze the current in detail, our findings provide strong evidence that I(Na) is present in human sinoatrial node pacemaker cells, and that this I(Na) is functionally available at potentials negative to -60 mV.

Abstract 1509: Inducible TBX3 Overexpression As A Tool For Biopacemaker Engineering

Introduction: Currently constructed biopacemakers based on single gene transfer strategies function suboptimally, with periods of slow heart rates and instabilities during rest. In the sinoatrial node (SAN), the dominant native pacemaker, multiple genes are required for proper impulse formation and impulse propagation. TBX3 is an important regulator of the SAN gene program during heart development. We examined the effects of inducible TBX3 overexpression in adult hearts and in vitro we explored whether lentiviral TBX3 overexpression may be used in biopacemaker engineering. Methods: In vivo atrial and ventricular expression levels of the connexin isoforms Cx43 and Cx40 (impulse propagation) and SCN5A were studied in mice with tamoxifen inducible overexpression of TBX3 using quantitative PCR analysis. Single neonatal rat cardiac myocytes were transduced with TBX3 expressing lentivirus to analyze the effects of TBX3 on action potentials and membrane currents (impulse formation) using the perforated patch-clamp technique. Results: In vivo, Cx43, Cx40 and SCN5A, which are not or only moderately expressed in the native SAN, were severely down-regulated to 20%, 15%, and 40%, respectively, by TBX3 (n=12; p<0.01). Single neonatal cardiac myocytes overexpressing TBX3 exhibited faster spontaneous beating rates, along with decreased maximum diastolic potential, inward rectifier potassium current (I K1 ), and fast sodium current (I Na ). These properties are typical of SAN pacemaker cells. Conclusions: TBX3 can act as a strong repressor of the working myocardium gene program in the adult heart. Overexpression of TBX3 might be a useful tool in biopacemaker gene and cell therapy.

Fibroblasts: modulating the rhythm of the heart

Cardiac fibroblasts have been implicated in arrhythmia initiation and maintenance affecting electrical propagation through slow, discontinuous conduction. Fibroblasts are unexcitable cells, their resting membrane potential is more depolarized than that of myocytes and their membrane resistance is higher. These characteristics suggest that, if coupled to myocytes, fibroblasts may function as current sinks and/or sources for electrical charge and as shortand long-range conductors. Abnormal accumulation of fibroblasts occurs in the heart under pathological conditions and ageing, which may alter normal cardiac function. Most clinical, experimental and numerical studies have regarded fibroblasts and fibrosis as electrically insulating obstacles. However, recent suggest that when coupled to cardiac myocytes, fibroblasts could influence impulse propagation not only by acting as passive transducers but also by influencing excitability of neighbouring myocytes (Miragoli et al. 2007). Compared to the atria and ventricles, the sinoatrial (SA) node contains a relatively higher amount of fibroblasts, which can be electrically coupled to pacemaker cells. Electrical coupling through gap junctions in the SA node is important for achieving pacemaker synchronization (Jalife, 1984). Pacemaker cells in the intact SA node beat at a common frequency but when isolated they show large variations in intrinsic beating frequency. Functional electrical coupling of SA nodal pacemaker cells through connexins forming gap junctions has been demonstrated in several species. However, the connexin isotypes expressed in the SA node vary somewhat depending on the species. Cx43, the most abundant connexin in the heart, has also been found in the SA node of rabbit, hamster and guinea pig although it is less abundant than other connexins. Interestingly, Cx43 was not detected in rat, cow or human nodal tissue. Recently, Kreuzberg et al. (2005) demonstrated that the mouse SA node expresses Cx30.2. Further studies using Cx30.2-knock-out mice suggest that Cx30.2 provides higher intercellular resistance and slower conductance due to a relatively small unitary conductance (∼9 pS). Nevertheless, the role of gap junctions in heterocellular tissue in the SA node remains unknown. The study by Fahrenbach et al. (2007) provides indirect evidence toward addressing this question. They investigated the mechanism through which non-excitable cells influence the spontaneous activity of pacemaker cells using heterocellular monolayers of HL-1 cells, a cell line derived from murine atrial myocyte tumour lineage AT-1, and fibroblasts that were isolated from neonatal rat and mouse ventricular tissue. The influence of fibroblasts on frequency of spontaneous activity, conduction velocity and interbeat interval variability were studied by co-culturing fibroblasts and myocytes at varying ratios.

Ionic Mechanisms Underlying the Positive Chronotropy Induced by β1-Adrenergic Stimulation in Guinea Pig Sinoatrial Node Cells: a Simulation Study

Positive chronotropy induced by beta1-adrenergic stimulation is achieved by multiple interactions of ion channels and transporters in sinoatrial node pacemaker cells (SANs). To investigate the ionic mechanisms, we updated our SAN model developed in 2003 and incorporated the beta1-adrenergic signaling cascade developed by Kuzumoto et al. (2007). Since the slow component of the delayed rectifier K+ current (IKs) is one of the major targets of the beta1-adrenergic cascade, we developed a guinea pig model with a large IKs. The new model provided a good representation of the experimental characteristics of SANs. A comparison of individual current during diastole recorded before and after beta1-adrenergic stimulation clearly showed the negative shift of the L-type Ca2+ current (ICaL) takeoff potential, enlargement of the sustained inward current (I st), and the hyperpolarization-activated nonselective cation current (Iha) played major roles in increasing the firing frequency. Deactivation of IKs during diastole scarcely contributed to the time-dependent decrease in membrane K+ conductance, which was the major mechanism for slow diastolic depolarization, as indicated by calculating the instantaneous equilibrium potential (lead potential). This was because the activation of IKs during the preceding action potential was negligibly small. However, IKs was important in counterbalancing the increase in ICaL and the Na+/Ca2+ exchange current (INaCa), which otherwise compromised the positive chronotropic effect by elongating the action potential duration. Enhanced Ca2+ release from the sarcoplasmic reticulum failed to induce an obvious chronotropic effect in our model.

Effects of Intracellular Ca2+ Dynamics on Bifurcation Structures of Sinoatrial Node Pacemaker Cells: a theoretical study

Effects of Intracellular Ca2+ Dynamics on Bifurcation Structures of Sinoatrial Node Pacemaker Cells: a theoretical study

Electrophysiology of hyperpolarization-activated cyclic nucleotidegated cation channel 2 and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 expressed in HEK293 cells

Electrophysiology of hyperpolarization-activated cyclic nucleotidegated cation channel 2 and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 expressed in HEK293 cells

Abstract 488: Sinoatrial Nodal Pacemaker Cells (SANC) Exhibit High Basal CA Activated Adenylyl Cyclase (AC) Activity and Express Multiple Distinctly Localized AC Types

In SANC constituitive AC generates high basal cAMP, inducing PKA-dependent phosphorylation that regulates Ca2+ cycling, that is essential for normal pacemaker function. Our goals were to identify, in rabbit SANC, the types of AC expressed, and their Ca2+ sensitivity and location. Radioimmunoassay (with total phosphodiasterase inhibition) showed a high Ca2+ activated basal AC activity. AC activity increased 5-fold from Ca2+ free (EGTA) to 1 uM free Ca2+. RT PCR (using specifically designed rabbit primers) showed that AC types II and V, and Ca2+ activated types, I and VIII, are expressed in SANC. The organization of these distinct AC types within calveolar or non-calveolar membrane microdomains was determined in pooled SANC isolated from 5 hearts, using triton x100, and sucrose gradient ultracentrifugation. Lipid domains segregated into caveolin containing and non-caveolin containing membrane microdomains, where AC activity was concentrated (fig , AC activity). Immunoblots demonstrated localization of different AC types between these two membrane domains, with AC I, II, V/VI localizing to caveolin containing lipid rafts, and AC VIII present in both caveolin and GM1 lipid domains, and also in the soluble fraction (fig ). In summary, multiple ACs, both Ca2+ activated and non-CA2+ activated types, are expressed in SANC, and these reside in distinct calveolar and non-calveolar lipid domains. We conclude that constituitive basal AC activity is, generated, in part, at least, by a Ca2+ activated AC. type.