Rabbit Sinus Node Research Articles

The reverse mode of the Na+/Ca2+ exchanger contributes to the pacemaker mechanism in rabbit sinus node cells

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca2+-handling. The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 μM ORM-10962. Mechanistic simulations were performed by Maltsev–Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 μM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.

Adherens junction engagement regulates functional patterning of the cardiac pacemaker cell lineage.

Cardiac pacemaker cells (CPCs) rhythmically initiate the electrical impulses that drive heart contraction. CPCs display the highest rate of spontaneous depolarization in the heart despite being subjected to inhibitory electrochemical conditions that should theoretically suppress their activity. While several models have been proposed to explain this apparent paradox, the actual molecular mechanisms that allow CPCs to overcome electrogenic barriers to their function remain poorly understood. Here, we have traced CPC development at single-cell resolution and uncovered a series of cytoarchitectural patterning events that are critical for proper pacemaking. Specifically, our data reveal that CPCs dynamically modulate adherens junction (AJ) engagement to control characteristics including surface area, volume, and gap junctional coupling. This allows CPCs to adopt a structural configuration that supports their overall excitability. Thus, our data have identified a direct role for local cellular mechanics in patterning critical morphological features that are necessary for CPC electrical activity.

Novel Na+/Ca2+ Exchanger Inhibitor ORM-10962 Supports Coupled Function of Funny-Current and Na+/Ca2+ Exchanger in Pacemaking of Rabbit Sinus Node Tissue.

Background and PurposeThe exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca2+ handling and sarcolemmal ion channels. An important player of this crosstalk is the Na+/Ca2+ exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the If-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN).Experimental ApproachCurrents were measured by patch-clamp, Ca2+-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv et al. SAN model.Key ResultsORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca2+ level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (If) was previously inhibited and vice versa, the effect of If was augmented when the Ca2+ handling was suppressed.Conclusion and ImplicationsWe confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between If and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of If or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking.

Implication of the TRPM4 nonselective cation channel in mammalian sinus rhythm

The transient receptor potential melastatin 4 (TRPM4) channel is expressed in the sinoatrial node, but its physiologic roles in this tissue with cardiac pacemaker properties remain unknown. This Ca(2+)-activated nonselective cation channel (NSCCa) induces cell depolarization at negative potentials. It is implicated in burst generation in neurons and participates in induction of ectopic beating in cardiac ventricular preparations submitted to hypoxia/reoxygenation. Accordingly, TRPM4 may participate in action potential (AP) triggering in the sinoatrial node. The purpose of this study was to investigate the influence of TRPM4 on spontaneous heart beating. Spontaneous APs were recorded using intracellular microelectrodes in mouse, rat, and rabbit isolated right atria. In the spontaneously beating mouse atrium, superfusion of the TRPM4-specific inhibitor 9-phenanthrol produced a concentration-dependent reduction in AP rate (maximal reduction = 62% that of control; EC50 = 8 × 10(-6) mol●L(-1)) without affecting other AP parameters. These effects were absent in TRPM4(-/-) mice. 9-Phenanthrol exerted a rate-dependent reduction with a higher effect at low rates. Similar results were obtained in rat. Moreover, application of 9-phenanthrol produced a reduction in diastolic depolarization slope in rabbit sinus node pacemaker cells. These data showed that TRPM4 modulates beating rate. Pacemaker activity in the sinoatrial node results from the slow diastolic depolarization slope due to the "funny" current, Na/Ca exchange, and a Ca(2+)-activated nonselective cation current, which can be attributable in part to TRPM4 that may act against bradycardia.

Electrophysiological mechanisms of sophocarpine as a potential antiarrhythmic agent

To examine the electrophysiological effects of sophocarpine on action potentials (AP) and ionic currents of cardiac myocytes and to compare some of these effects with those of amiodarone. Langendorff perfusion set-up was used in isolated guinea pig heart, and responses to sophocarpine were monitored using electrocardiograph. Conventional microelectrode, voltage clamp technique and perforated patch were employed to record fast response AP (fAP), slow response AP (sAP) and ionic currents in guinea pig papillary muscle or rabbit sinus node cells. Tachyarrhythmia produced by isoprenaline (15 μmol/L) could be reversed by sophocarpine (300 μmol/L). Sophocarpine (10 μmol/L) decreased the amplitude by 4.0%, maximal depolarization velocity (V(max)) of the fAP by 24.4%, and Na(+) current (I(Na)) by 18.0%, while it prolonged the effective refractory period (ERP) by 21.1%. The same concentration of sophocarpine could also decrease the amplitude and V(max) of the sAP, by 26.8% and 25.7%, respectively, and attenuated the Ca(2+) current (I(CaL)) and the K(+) tail current substantially. Comparison of sophocarpine with amiodarone demonstrated that both prolonged the duration and the ERP of fAP and sAP, both decreased the amplitude and V(max) of the fAP and sAP, and both slowed the automatic heart rate. Sophocarpine could reverse isoprenaline-induced arrhythmia and inhibit I(Na), I(CaL), and I(Kr) currents. The electrophysiological effects of sophocarpine are similar to those of amiodarone, which might be regarded as a prospective antiarrhythmic agent.

Effects of S(+)-efonidipine on the rabbit sinus node action potential and calcium channel subunits Ca V1.2, Ca V1.3 and Ca V3.1

The effect of S(+)-efonidipine on sinus node action potential and calcium channel α-subunits was examined. The slope of the phase 4 depolarization of isolated rabbit sinus node tissue was significantly reduced by S(+)-efonidipine (1 μM), slightly reduced by nifedipine (1 μM), but was not affected by R(−)-efonidipine. S(+)-efonidipine (1 μM), inhibited the expressed Ca V1.2, Ca V1.3 and Ca V3.1 channel currents by 75.7%, 75.3% and 94.0%, nifedipine 84.0%, 43.2% and 14.9%, and R(−)-efonidipine 30.0%, 19.6% and 92.8%, respectively. Thus, the prolongation of the phase 4 depolarization of the rabbit sinus node by S(+)-efonidipine may be explained by blockade of the Ca V1.3 channel current.

The Heart Rate‐Lowering Agent Ivabradine Inhibits the Pacemaker Current If in Human Atrial Myocytes

It has been speculated that pacemaker current (I(f)) in human atria could play a role in causing ectopic atrial automaticity. Ivabradine is a novel selective and specific I(f) inhibitor in the sinus node that reduces heart rate without any negative inotropic effect. The aim of the study was to explore possible effects of ivabradine on I(f) in atrial myocytes. Using patch-clamp technique, we studied effects of ivabradine on I(f) present in atrial myocytes isolated from human right appendages of patients undergoing cardiac surgery. The identification of HCN isoforms was obtained by means of multiplex single-cell RT-PCR. Ivabradine induced a marked concentration and use-dependent I(f) inhibition with an IC50 at steady state of 2.9 microM. Time constant of block development (Tau(on)) decreases with the increase in the ivabradine concentration. Use-dependent inhibition induced by ivabradine (3 microM) was not modified in the presence of cAMP (10 microM) in the pipette solution. Multiplex single-cell RT-PCR indicates that the major HCN gene subtype detected in atria was HCN2. HCN4 is detected weakly and HCN1 is not significantly detected. Ivabradine inhibits I(f) current in the nonpacemaker cell with characteristics similar to those described previously in rabbit sinus node cells, but revealed a lesser sensitivity for I(f) recorded in human atrial cell than hHCN4 subunits considered as the major contributors to native f-channels in human sinoatrial node. A potential protection of atrial arrhythmias by ivabradine is discussed.

Acute In Vitro Effects of Dronedarone, an Iodine-Free Derivative, and Amiodarone, on the Rabbit Sinoatrial Node Automaticity: A Comparative Study

Amiodarone is a potent antiarrhythmic drug commonly used in the treatment of supraventricular and ventricular arrhythmias. Dronedarone is a recently developed iodine-free compound (Sanofi Recherche), structurally related to amiodarone. Amiodarone and dronedarone have shown similar long-term effects on sinoatrial node automaticity in vivo and in vitro in the rabbit heart. In the present study, we used a microelectrode technique to compare the acute in vitro electrophysiologic effects of amiodarone (100 microM) and dronedarone (100 microM) on the rabbit sinus node. Like amiodarone, dronedarone induces a marked reduction in sinus node automaticity, evidenced by decreases in spontaneous beating rate, action potential amplitude, and slope of phase 4 depolarization. Isoproterenol dose-dependently increases sinus node automaticity in the presence of either amiodarone or dronedarone. The data suggest that dronedarone may be a useful antiarrhythmic alternative to amiodarone in the treatment of supraventricular arrhythmias.

Variety is the spice of life: searching for the substrates of regional myocardial electrical properties

Variety and diversity as well as regional specificity of gene expression are the basis of normal cardiac function. Transcriptional regulation has been demonstrated to be an essential compensatory response to structural and functional changes in the heart. In order to understand transcriptional regulation in defined disease states it is essential to have a detailed knowledge of expression patterns in non-diseased myocardial tissue. In this issue of The Journal of PhysiologyGaborit et al. (2007) give a detailed analysis of differentially expressed ion channel genes controlling regional diversity of myocardial electrical properties in non-diseased human hearts. High-throughput real-time RT-PCR in a hypothesis driven approach allowed for a comprehensive description of 79 ion channels and related genes in specific regions of the human heart. Differential expression of a variety of genes is giving clues to the molecular substrates controlling distinct myocardial electrical properties of specific regions in the heart and has been demonstrated in anatomically distinct regions of the heart such as atrial versus ventricular myocardium (Barth et al. 2005) or ventricular endocardium versus ventricular epicardium (Rosati et al. 2001). Genome wide expression studies in heart failure resulted in a description of deregulated gene clusters that can be attributed and sorted by functional groups such as metabolism, inflammation and signal transduction. While regional differences in deregulation of ion channels in normal and failing heart has been noted (Ellinghaus et al. 2005; Kaab et al. 2004), this study, in an unprecedented way, investigates a comprehensive set of ion channel genes and related genes in atria, ventricular epicardium and endocardium as well as in Purkinje fibres. The authors present and discuss their data in the context of previous findings and highlight novel findings of potential functional significance. Among the wealth of data, the detailed analysis of the Purkinje fibre system deserves special attention. The participation of Nav1.7 in TTX-sensitive Purkinje fibre INa, and the importance of low-level Na+,K+-ATPase and high-level inositol-trisphosphate receptor expression in the Purkinje system warrant further functional studies with respect to arrhythmogenesis. These data add to the discussion on Ca2+ signalling in the Purkinje system that was substantiated by the recent discovery of the molecular substrates of T- and L-type Ca2+ currents in Purkinje fibres in dog (Rosati et al. 2007). In comparison to previous pan-genomic approaches on regional specific gene expression in the heart this quantitative RT-PCR based study allowed for the detection of low-abundance mRNAs and unmasked differences in expression levels to a greater extent than pan-genomic arrays (Kaab et al. 2004; Ellinghaus et al. 2005). These data provide an important reference with a focus on low abundance ion channel genes for other studies aiming at: genes exclusively expressed in atrial or ventricular tissue; genes controlling electrical properties that may play an important functional role in arrhythmogenesis; transcriptional deregulation contributing to a remodelling process in defined disease states in the human heart (e.g. hypertrophy, heart failure). To link deregulation of ion channel genes to other potentially more global gene deregulation processes in the course of specific disease states more information especially on transcription factors involved will be needed. More sophisticated and meticulous dissecting methods as well as a combination with functional studies have recently led to the detailed characterization of ion channel expression in rabbit atria and sinus node, linking the variety of gene expression on a microscopic level to specific electrical properties that guarantee physiological excitation and conduction (Tellez et al. 2006). This holds out the promise that we will see a detailed analysis and understanding of the transcriptional control of key anatomical substrates of excitation and conduction such as sinus node, atrio-ventricular node, and crista terminalis in the human heart in the future.

Study of the Preautomatic Pause under Exposure to Acetylcholine in True Pacemaker Cells of Rabbit Sinus Node Using Computer Simulation

Study of the Preautomatic Pause under Exposure to Acetylcholine in True Pacemaker Cells of Rabbit Sinus Node Using Computer Simulation

Study of the Effect of Acetylcholine on Intracellular Homeostasis of True Pacemaker Cells of Rabbit Sinus Node Using Computer Simulation

Study of the Effect of Acetylcholine on Intracellular Homeostasis of True Pacemaker Cells of Rabbit Sinus Node Using Computer Simulation

Study of the Effect of Acetylcholine on the Excitability of True Pacemaker Cells of Rabbit Sinus Node Using Computer Simulation

Study of the Effect of Acetylcholine on the Excitability of True Pacemaker Cells of Rabbit Sinus Node Using Computer Simulation

Sélection et caractérisation pharmacologique de Procoralan®, un inhibiteur sélectif du courant pacemaker If

The screening of a series of benzocycloalkane derivatives led to the selection of Procoralan® (ivabradine), the first selective inhibitor of the depolarizing If (funny) current of the sinus node, for the treament of myocardial ischaemia. In vitro, this compound reduces the spontaneous beating rate of isolated right rat atria and the firing rate of the action potential of rabbit sinus node preparations. This effect is explained by a reduction in the diastolic depolarisation slope of the action potential and underlies a selective inhibition of the pacemaker If current. In vivo, it induces a selective reduction in heart rate both at rest and during exercise. It preserves myocardial contractility, atrioventricular conduction and ventricular repolarisation duration. Ivabradine exerts a similar anti-ischaemic activity in exercise-induced myocardial ischaemia in pigs to that of a β-blocker and, furthermore, it limits to a greater extent ischaemic myocardial contractile dysfunction.

Study of the Effect of Acetylcholine on Ion Currents in Single Cells of True and Latent Pacemakers of Rabbit Sinus Node using Computer Simulation

Study of the Effect of Acetylcholine on Ion Currents in Single Cells of True and Latent Pacemakers of Rabbit Sinus Node using Computer Simulation

Estimating the active dimension of cardiac pacemaking dynamics

Although there exist several different models for the excitability of cardiac and nerve cells, the number of ionic currents that determine this excitability is not clearly known. On the other hand, the chaotic behavior of excitable cells has recently been demonstrated. The science of complex dynamics provides tools for the reconstruction of the original dynamics from a time series of observed data. We determined the degrees of freedom for the excitability of a single cardiac pacemaker cell by chaotic analysis of a time series of action potentials (APs) recorded from the rabbit sinus node. We provide evidence that at least 12 variables are needed to determine the AP. From our findings, we conclude that a new model for the cardiac pacemaker mechanism may be needed, because previous models could deal only with fewer than 12 variables. Our approach based on our observation of a time series offers a powerful tool for constructing a model for the underlying biological mechanism.

Morphological and membrane characteristics of spider and spindle cells isolated from rabbit sinus node.

This study reports the comparative quantitative, morphological, and electrophysiological properties of two pacemaker cell types, spider and spindle-shaped cells, isolated from the rabbit sinoatrial node. Isolated nodal cells were studied with perforated and ruptured patch whole cell recording techniques. The basic spontaneous cycle length of the spider cells was 381 +/- 12 ms, and the basic spontaneous cycle length of the spindle cells was 456 +/- 17 ms (n = 12, P < 0.05). The spider cells had a more positive maximum diastolic potential (-54 +/- 1 mV) compared with the spindle cells (-68 +/- 1mV, P < 0.05). The overshoot and action potential amplitudes were also smaller in the spider cells. The hyperpolarization-activated inward (I(f)) current density, measured from their tail currents, was 15 +/- 1.3 pA/pF for the spider cells and 9 +/- 0.7 pA/pF for the spindle cells (P < 0.01). I(f) current activation voltage was more positive in the spider cells than the spindle cells. Isoproterenol (1 microM) decreased the spontaneous cycle length of the spider cells by 28 +/- 3% and the spindle cells by 20 +/- 1.5% (P < 0.05). Acetylcholine (0.5 microM) hyperpolarized the membrane potential of the spider cells to -86 +/- 0.7 mV and the spindle cells to -76 +/- 0.8 mV (P < 0.05). In summary, there are at least two distinct pacemaker cell types in the sinus node with different electrophysiological characteristics.

Mibefradil, an ICa,T blocker, effectively blocks ICa,L in rabbit sinus node cells

To test the hypothesis that the Ca 2+ channel blocker mibefradil slows heart rate due to inhibition of T-type Ca 2+ current in pacemaker cells, we studied effects of mibefradil on action potentials and ionic currents of isolated rabbit sinus node cells using the patch clamp technique. Mibefradil (100 nM and 1 μM) reduced spontaneous rate, decreased action potential amplitude and finally stopped impulse initiation. This action was not due to the drug effect on hyperpolarization-activated pacemaker current, but can be explained by attenuation of both T- and L-type Ca 2+ currents, which were inhibited by mibefradil almost equally (55% and 64% inhibition with 1 μM for T- and L-types, respectively).

Positive chronotropic actions of parathyroid hormone and parathyroid hormone-related peptide are associated with increases in the current, I(f), and the slope of the pacemaker potential.

The classic calciotropic hormone parathyroid hormone (PTH) and its paracrine factor parathyroid hormone-related protein (PTHrP) both increase heart rate. We used standard electrophysiological techniques to study the effects of PTH and PTHrP on isolated rabbit sinus node, isolated canine Purkinje fibers, and disaggregated rabbit sinus node myocytes. Sinus node maximum diastolic potential, activation voltage, and amplitude were unchanged by PTH or PTHrP (P>.05). However, the slope of phase 4 and the automatic rate were increased at PTH and PTHrP > or = 10 nmol/L (P<.05). Comparable results were seen in canine Purkinje fibers. We then used the perforated-patch technique to study the I(f) pacemaker current in sinus node. PTH 12.5 nmol/L and PTHrP 12.5 to 18 nmol/L increased I(f) at -65 mV by 68+/-41% (n=5) and 69+/-50% (n=5), respectively. Actions of both agents were reversible. The increase in I(f) appeared to result from a change in maximal conductance and not a shift in the voltage dependence of activation. These observations provide, for the first time, direct electrophysiological support for the chronotropic actions of PTH and PTHrP. They suggest that classic hormones and paracrine factors can have multiple functions and that in the case of PTH and PTHrP, a newly recognized action is to alter automaticity directly.

Effects of potassium channel openers on Na + and K + currents in rabbit sinus node and atrial myocytes

The effects of the potassium channel openers (KCOs) Cromakalim and Lemakalim on rabbit sinoatrial and atrial myocytes were examined by means of the whole-cell patch-clamp technique. Lemakalim (up to 100 μM) had no effect on potassium current in sinoatrial cells. Both Lemakalim and Cromakalim (100 μM) displayed a two-fold action on atrial myocytes: (1) they increased an outwardly rectifying conductance at potentials positive to E K and, (2) they markedly decreased a TTX-sensitive Na + current active in the voltage range −50 −30 mV. This novel action on TTX-sensitive currents is of particular since these two benzopyrans have been thought to specifically target potassium channels.

Gap junctional channels in adult mammalian sinus nodal cells. Immunolocalization and electrophysiology.

The subcellular mechanism of cell-to-cell communication in the natural pacemaker region of the mammalian heart was studied using electrophysiological and immunofluorescence techniques in isolated pairs of rabbit sinus nodal cells. By measuring whole-cell currents using a double patch-clamp approach, it was demonstrated that communication in the sinus node is mediated through gap junctional channels similar to those in other types of adult cardiac cell pairs. Macroscopic sinus nodal junctional resistance had a mean value of 387.9 +/- 97.1 M omega (mean +/- SEM, n = 10) and was greatly increased by superfusion with alkanols. Single-channel junctional conductance could be resolved in three cell pairs. Given their high membrane resistance (1.16 +/- 0.32 G omega, n = 12), the electrical coupling provided by as few as three gap junctional channels between nodal cells will allow for pacemaker synchronization. Further evidence for the presence of the channels was obtained from immunofluorescent double-labeling of desmin and the gap junction protein (connexin43) in sinus nodal tissue as well as in cultured sinus nodal cells. Using antisera against residues 243-257 of the connexin43 protein, a specific staining at the site of cell-to-cell apposition was demonstrated. These data provide direct evidence in favor of electronic coupling as the means for achieving pacemaker synchronization in the rabbit sinus node.