Rabbit Sinoatrial Node Pacemaker Cells Research Articles

How “Funny” is the Cardiac Pacemaking? a Quantitative Analysis Based on Dynamic Clamp Recordings

A typical feature of sinoatrial (SA) node pacemaker cells is the presence of an ionic current that activates upon hyperpolarization. The quantitative role of this hyperpolarization-activated current known as the 'funny current', If, in the spontaneous pacemaker activity of SA nodal cells remains a matter of intense debate.We aimed to characterize the impact of If as implemented in recent conflicting mathematical models of rabbit SA node pacemaker cells by using the Dynamic Clamp technique.Experiments were performed using the Real-Time eXperiment Interface (RTXI) which is a real-time biological experimentation system based on Real-Time Linux. The time course of If was dynamically reconstructed from the experimentally recorded action potentials of a single isolated rabbit SA nodal pacemaker cell according to the mathematical models of Maltsev and Lakatta(2009) and Severi et al.(2012). Two different protocols were implemented: i) selective If block by Ivabradine (3μM) and substitution with the syntethic If ii) comparison of the effects of a virtual Isoprenaline(ISO)-induced shift (7.5 mV) of If activation with the real effects of 1μM ISO.In relation to the substitution of the Ivabradine-sensitive current, the model of Severi et al. (Sev) allowed to restore the control pacemaking rate (Ctrl [Hz]: 2.77±0.13 Vs Sev: 2.70±0.09, N.S.), whereas Maltsev and Lakatta's (ML) induced only a smaller recovery from current-blocking conditions (Control [Hz]: 2.77±0.13 vs ML: 2.39±0.10, p<0.05). Preliminary results on the virtual ISO-induced shift produced a rate increase comparable to the real drug effect (+42±9 % vs +58±15 %, N.S.), suggesting that If accounts for most of the ISO-induced rate increase.Dynamic Clamp results are in agreement with the quantitative description of the If impact on the modulation of cardiac pacemaking provided by our recent computational sinoatrial model (Severi et al. 2012).

Calcium Transient and Sodium‐Calcium Exchange Current in Human versus Rabbit Sinoatrial Node Pacemaker Cells

There is an ongoing debate on the mechanism underlying the pacemaker activity of sinoatrial node (SAN) cells, focusing on the relative importance of the “membrane clock” and the “Ca2+ clock” in the generation of the small net membrane current that depolarizes the cell towards the action potential threshold. Specifically, the debate centers around the question whether the membrane clock-driven hyperpolarization-activated current, I f, which is also known as the “funny current” or “pacemaker current,” or the Ca2+ clock-driven sodium-calcium exchange current, I NaCa, is the main contributor to diastolic depolarization. In our contribution to this journal's “Special Issue on Cardiac Electrophysiology,” we present a numerical reconstruction of I f and I NaCa in isolated rabbit and human SAN pacemaker cells based on experimental data on action potentials, I f, and intracellular calcium concentration ([Ca2+]i) that we have acquired from these cells. The human SAN pacemaker cells have a smaller I f, a weaker [Ca2+]i transient, and a smaller I NaCa than the rabbit cells. However, when compared to the diastolic net membrane current, I NaCa is of similar size in human and rabbit SAN pacemaker cells, whereas I f is smaller in human than in rabbit cells.

Ethanol Enhances Human Hyperpolarization‐Activated Cyclic Nucleotide‐Gated Currents

There is a clear association between excessive ethanol ( EtOH ) consumption and the risk of sudden cardiac death. The hyperpolarization-activated cyclic nucleotide-gated (HCN) current, I (f) , is known to contribute to spontaneous pacemaker activity of sinoatrial (SA) node cells. However, the exact mechanisms of EtOH on arrhythmia induction are not well understood. The preparations of SA node were excised from rabbit heart, transmembrane potentials were recorded by standard glass microelectrode technique, and a whole-cell patch clamp technique was used to record I (f) in enzymatically isolated rabbit SA node pacemaker cells. Human HCN2 (hHCN2) and HCN4 channels were heterologously expressed in Xenopus oocytes and studied using 2-electrode voltage clamp technique. Superfusion of EtOH increased the spontaneous firing frequency of SA node cells in a reversible fashion. Treatment with ivabradine irreversibly depressed basal firing frequency and markedly attenuated the enhancement effect of EtOH on firing. The stimulatory effects of EtOH on I (f) were concentration-dependent in the range of 1 to 100mM, with an average EC (50) value of 20.81±6.71mM and Hill coefficient of 1.19±0.10. Furthermore, EtOH reversibly enhanced the HCN currents in a concentration-dependent fashion with an EC (50) value of 18.41±2.75 mM for the HCN2 channel and 21.98±3.54mM for the HCN4 channel, which was accompanied by the acceleration of activation and deactivation kinetics. In addition, EtOH , at both moderate and high doses, caused a shift in the voltage dependence of hHCN4 channel activation to more depolarizing potentials. However, superfusion of high, not moderate, concentration of EtOH caused a shift in the voltage dependence of hHCN2 channel activation to more hyperpolarizing potentials. This study provides insight into the molecular interaction of EtOH and the hHCN channels, which may shed light on elucidating the potentially proarrhythmic mechanism of EtOH .

Blocking effects of acehytisine on pacemaker currents (If) in sinoatrial node cells and human HCN4 channels expressed in Xenopus laevis oocytes

Ethnopharmacological relevanceThe root of Aconitum coreanum (Levl.) Raipaics has been extensively used to treat various kinds of disorders including cardiovascular disease in China for a long time. According to recent studies, its antiarrhythmic actions are attributable to the active component, acehytisine. However, the underlying mechanism remains poorly understood. Aim of the studyThe effects of acehytisine on the spontaneous activity in sinoatrial nodes and the electropharmacological action of this drug on If in pacemaker cells and hHCN4 channels in oocytes were to be investigated. Materials and methodsSinoatrial nodes were cut from rabbit heart, and transmembrane potentials were recorded by standard microelectrode technique. A whole-cell patch clamp technique was employed to record If isolated enzymatically from rabbit sinoatrial node pacemaker cells. Human HCN4 channels were heterologously expressed in Xenopus oocytes and studied using the two-electrode voltage clamp technique. ResultsAcehytisine decreased the pacemaker rate of firing and slope of diastolic depolarization, modified the action potential configurations and blocked If in rabbit sinoatrial node cells and hHCN4 channels expressed in Xenopus oocytes in a concentration-dependent, voltage-independent and non-use-dependent manner. Its electropharmacological properties were consistent with those of a close-state blocker. ConclusionOur findings are likely to shed light on the clinical application of acehytisine in the treatment of cardiovascular disorders.

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.

Non‐Muscarinic and Non‐Nicotinic Inhibition by the Acetylcholine Analogue Carbachol of the Delayed Rectifier Potassium Current, ik, in Rabbit Isolated Sino‐Atrial Node Cells

The effect of carbachol, an analogue of acetylcholine, on the delayed rectifier potassium current, iK, was investigated in rabbit isolated sino-atrial node cells using the whole cell patch clamp technique with amphotericin-permeabilized patches. In the presence of 500 nM atropine and 500 nM hexamethonium to block muscarinic and nicotinic receptors, respectively, 500 nM carbachol decreased the amplitude and rate of deactivation of iK without, however, affecting the slope of the iK activation curve. The same concentration of carbachol decreased the pacemaking rate of spontaneously active sino-atrial node cells by more than 13%. Thus, there is a non-muscarinic and non-nicotinic pathway for cholinergically induced reduction in the amplitude and rate of deactivation of iK that would appear to contribute to negative chronotropy in rabbit sinoatrial node pacemaker cells.

The sustained inward current in sino-atrial node cells of guinea-pig heart.

Single myocytes were dissociated from the sino-atrial (SA) node of guinea-pig hearts. Only a quite small fraction of the cell population showed spontaneous action potentials and these cells were characterized by the presence of the hyperpolarization-activated cation current If , the delayed rectifier K+ current IK and the L-type Ca2+ current ICa,L as well as by the absence of both the transient outward current Ito and the inward rectifier K+ current IK, 1. After blocking If and IK, depolarizing pulses from -80 mV revealed a large nicardipine-sensitive late current (NSLC). The NSLC was scarcely affected by decreasing extracellular [Ca2+] ([Ca2+]o) from 1.8 to 0.1 mM, while it was decreased significantly by depleting [Na+]o, differently from ICa,L. NSLC was blocked by nicardipine and was increased by Bay K 8644. NSLC was increased by isoprenaline and the additional application of acetylcholine reversed the increase of this current. We conclude that NSLC is largely composed of Ist described in the rabbit SA node pacemaker cells, and that Ist is unique for the pacemaker cells in mammalian SA node cells. Most of the quiescent cells showed neither If nor Ist.

Existence of a low-threshold and sustained inward current in rabbit atrio-ventricular node cells.

Single myocytes were dissociated from the atrio-ventricular (AV) node of rabbit hearts. Spontaneous action potentials of the N or NH type were recorded from the fusiform AV node cells. Under whole-cell voltage clamp, these cells were characterized by the presence of hyperpolarization-activated cation current (I(f)), delayed rectifier K+ current (IK), and L-type Ca2+ current (ICa,L). After blocking I(f) and IK, depolarizing pulses from -80 mV revealed a sustained inward current, which was suppressed by 1 microM nicardipine. The activation threshold of the sustained inward current was around -60 mV and the maximum amplitude was 25.4 +/- 20.1 pA at -40 mV (n = 15). The current was also blocked by 1 microM verapamil. The sustained inward current was not decreased (125 +/- 32% of control, N = 5) by reducing [Ca2+]o from 1.8 to 0.1 mM, while it was nearly suppressed by depleting [Na+]o, differently from ICa,L. We concluded that the nicardipine-sensitive sustained inward current is largely attributable to Ist described in the rabbit sino-atrial node pacemaker cells. I(f) and Ist were not found in rod-shape AV node cells.

Identification and properties of an ATP-sensitive K+ current in rabbit sino-atrial node pacemaker cells.

1. Single myocytes were isolated from rabbit sino-atrial (SA) node by enzymatic dissociation. Spontaneous pacemaker activity, whole-cell and single-channel currents were recorded under conditions known to modulate ATP-sensitive K+ (KATP) channels. 2. The KATP channel openers, cromakalim and pinacidil, slowed or abolished the pacemaker activity, and caused hyperpolarization of the maximum diastolic potential (MDP). Glibenclamide, a KATP channel blocker, reversed these effects. Cromakalim- and pinacidil-activated currents reversed near the potassium equilibrium potential, EK. Glibenclamide had no effect on the L-type calcium current, ICa(L), the hyperpolarization-activated inward current, If, or the delayed rectifier potassium current, IK. 3. Sodium cyanide, which inhibits mitochondrial ATP production, induced a macroscopic current that reversed near EK and was blocked by glibenclamide. 4. In excised, inside-out patches from SA node cells, single KATP channels showed a slope conductance of 52 +/- 8 pS (mean +/- S.D.) when measurements were made at negative voltages in symmetric, 140 mM K+. Channels from ventricular myocytes showed a somewhat larger slope conductance (70 +/- 5 pS). 5. Raising the intracellular ATP concentration caused a concentration-dependent reduction in the open probability of the KATP channels (IC50, 16 microM; Hill coefficient, approximately 1; at both pH 7.4 and 6.8). 6. In excised inside-out patches, cromakalim or pinacidil induced significant increases in KATP channel activity in the presence of 50 microM or 1 mM intracellular ATP. This channel activity was blocked by glibenclamide. 7. Our results suggest that sino-atrial node cells express a distinct isoform of KATP channel which may play an important role in pharmacological and pathophysiological modulation of pacemaker activity.

Effects of SR 44866, a Potassium Channel Opener, on Action Potentials of Rabbit, Guinea Pig, and Human Heart Fibers

The effects of various concentrations (3 x 10(-8) - 1 x 10(-5) M) of SR 44866, a K+ channel opener, on action potential (AP) characteristics were investigated in isolated rabbit sinoatrial node (SAN), rabbit Purkinje fibers, guinea pig ventricle, human atrium, and human papillary muscle. SR 44866 (up to 1 x 10(-5) M), like cromakalim and pinacidil, did not modify SAN AP and automaticity of the rabbit heart. In atrial, Purkinje and ventricular fibers of animal and human hearts, SR 44866 did not significantly change membrane resting potential, AP amplitude, or maximum rate of phase 0 (dV/dtmax). The main AP modifications induced by SR 44866 were concentration-dependent reductions in plateau amplitude and AP duration (APD): IC50 2 x 10(-7), 7 x 10(-7), 1.4 x 10(-6), 2.5 x 10(-6), and much greater than 10(-5) M for human atrium, human ventricle, guinea pig ventricle, rabbit Purkinje, and rabbit atrium, respectively. In isolated guinea pig heart, SR 44866 induced decreases in contractions (IC50 1.7 x 10(-6) M) and coronary perfusion pressure (CPP) (IC50 2.1 x 10(-8) M) with a very slight reduction (5% at 1 x 10(-6) M) in spontaneous heart rate (HR). Negative inotropic effect (guinea pig) and APD shortenings (guinea pigs and humans) of SR 44866 (1 x 10(-6) and 3.10(-6) M) were antagonized by glibenclamide (3 x 10(-7) to 3 x 10(-6) M), a specific blocker of cardiac K+ATP channels. The data support the hypothesis that SR 44866 activates ATP-sensitive K+ channels, which are present in the atria and ventricles of the human heart but not in pacemaker cells of rabbit SAN.