Atrioventricular Nodal Myocytes Research Articles

Characterization of L-type calcium channel activity in atrioventricular nodal myocytes from rats with streptozotocin-induced Diabetes mellitus.

Cardiovascular complications are common in patients with Diabetes mellitus (DM). In addition to changes in cardiac muscle inotropy, electrical abnormalities are also commonly observed in these patients. We have previously shown that spontaneous cellular electrical activity is altered in atrioventricular nodal (AVN) myocytes, isolated from the streptozotocin (STZ) rat model of type-1 DM. In this study, utilizing the same model, we have characterized the changes in L-type calcium channel activity in single AVN myocytes. Ionic currents were recorded from AVN myocytes isolated from the hearts of control rats and from those with STZ-induced diabetes. Patch-clamp recordings were used to assess the changes in cellular electrical activity in individual myocytes. Type-1 DM significantly altered the cellular characteristics of L-type calcium current. A reduction in peak ICaL density was observed, with no corresponding changes in the activation parameters of the current. L-type calcium channel current also exhibited faster time-dependent inactivation in AVN myocytes from diabetic rats. A negative shift in the voltage dependence of inactivation was also evident, and a slowing of restitution parameters. These findings demonstrate that experimentally induced type-1 DM significantly alters AVN L-type calcium channel cellular electrophysiology. These changes in ion channel activity may contribute to the abnormalities in cardiac electrical function that are associated with high mortality levels in patients with DM.

Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node

The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits ‘fade’ or desensitization in the AVN, as established for the heart’s primary pacemaker – the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37°C. 0.1–1μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0s and τs 76.9s at −120mV; 1μM ACh). 10nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6s at −120mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.

Streptozotocin-induced diabetes modulates action potentials and ion channel currents from the rat atrioventricular node.

The present study was conducted to evaluate whether experimentally induced type 1 diabetes results in alterations to atrioventricular nodal (AVN) electrophysiology at the cellular level. Spontaneous action potentials (APs) and ionic currents were recorded from AVN myocytes isolated from the hearts of control rats and from those with streptozotocin-induced diabetes. Perforated patch-clamp recordings were used to assess changes in cellular AP parameters and in ionic currents. Type 1 diabetes significantly increased AP duration, whilst reducing AP firing rate, upstroke velocity and rate of diastolic depolarization. The diabetes-induced changes in AP parameters were accompanied by a significant leftward shift in the zero current potential under voltage clamp, a reduction in peak L-type Ca(2+) current density and reduced amplitude of delayed rectifier and hyperpolarization-activated currents. These findings demonstrate that experimentally induced type 1 diabetes can lead to remodelling of AVN cellular electrophysiology.

Lamin A/C haploinsufficiency causes dilated cardiomyopathy and apoptosis-triggered cardiac conduction system disease

Mutations in the lamin A/C (LMNA) gene, which encodes nuclear membrane proteins, cause a variety of human conditions including dilated cardiomyopathy (DCM) with associated cardiac conduction system disease. To investigate mechanisms responsible for electrophysiologic and myocardial phenotypes caused by dominant human LMNA mutations, we performed longitudinal evaluations in heterozygous Lmna+/− mice. Despite one normal allele, Lmna+/− mice had 50% of normal cardiac lamin A/C levels and developed cardiac abnormalities. Conduction system function was normal in neonatal Lmna+/− mice but, by 4 weeks of age, atrioventricular (AV) nodal myocytes had abnormally shaped nuclei and active apoptosis. Telemetric and in vivo electrophysiologic studies in 10-week-old Lmna+/− mice showed AV conduction defects and both atrial and ventricular arrhythmias, analogous to those observed in humans with heterozygous LMNA mutations. Isolated myocytes from 12-month-old Lmna+/− mice exhibited impaired contractility. In vivo cardiac studies of aged Lmna+/− mice revealed DCM; in some mice this occurred without overt conduction system disease. However, neither histopathology nor serum CK levels indicated skeletal muscle pathology. These data demonstrate cardiac pathology due to heterozygous Lmna mutations reflecting a 50% reduction in lamin protein levels. Lamin haploinsufficiency caused early-onset programmed cell death of AV nodal myocytes and progressive electrophysiologic disease. While lamin haploinsufficiency was better tolerated by non-conducting myocytes, ultimately, these too succumbed to diminished lamin levels leading to dilated cardiomyopathy, which presumably arose independently from conduction system disease.

Contribution of I(K,ADO) to the negative dromotropic effect of adenosine.

Despite the pathophysiological and therapeutic significance of the negative dromotropic effect of adenosine, its underlying ionic mechanism, and specifically the role of the adenosine-activated K(+) current (I(K,ADO)) is not experimentally defined. Therefore, we studied the contribution of I(K,ADO) to the negative dromotropic effect of adenosine. Effects of adenosine on single atrioventricular nodal and left atrial myocytes from rabbits were studied using the whole cell configuration of the patch clamp technique. Complementary experiments were done in rabbit and guinea pig isolated hearts instrumented to measure the atrium-to-His bundle interval. In contrast to its effect in atrial myocytes, Ba(2+) selectively and completely blocked I(K,ADO) at membrane potentials from -70 to 0 mV in atrioventricular nodal myocytes and abolished the adenosine-induced leftward shift of the reversal membrane potential. Ba(2+) alone did not significantly prolong the A-H interval, but markedly attenuated the A-H interval prolongation caused by adenosine. In guinea pig heart, EC(50) values ( pD(2) +/- SEM) for adenosine-induced atrium-to-His bundle interval prolongation were 3.3 micromol/L (5.48 +/- 0.04) and 13.2 micromol/L (4.88 +/- 0.05, P < 0.001) in the absence and presence of Ba(2+), respectively. Despite species-dependent differences in sensitivities to adenosine (guinea pig > rabbit), the relative contribution of adenosine-activated K(+) current to the atrium-to-His bundle interval prolongation was nearly identical. In guinea pig hearts it ranged from 37.8 % (P = 0.013) to 72.5 % (P < 0.001) at 2 to 6 micromol/L adenosine, respectively. I(K,ADO) contributes significantly to the negative dromotropic effect of adenosine, but predominantly at relatively high concentrations of the nucleoside.

Antagonism of the Positive Dromotropic Effect of Isoproterenol by Adenosine: Role of Nitric Oxide, cGMP-dependent cAMP-phosphodiesterase and Protein Kinase G

A. Zima, A. E. Martynyuk, C. N. Seubert, T. E. Morey, C. Sumners, R. F. Cucchiara and D. M. Dennis. Antagonism of the Positive Dromotropic Effect of Isoproterenol by Adenosine: Role of Nitric Oxide, cGMP-dependent cAMP-phosphodiesterase and Protein Kinase G. Journal of Molecular and Cellular Cardiology (2000) 32, 1609–1619. We hypothesized that nitric oxide (NO) plays an important role in mediating the anti-adrenergic effect of adenosine on atrioventricular (AV) nodal conduction. In guinea-pig hearts instrumented for measurement of AV nodal conduction time (atrium-to-His bundle, A–H, interval), the NO synthase (NOS) inhibitor, l-NMMA (100 μ m), reversibly inhibited 80% (P=0.009, n=6) of adenosine's anti-adrenergic action on the positive dromotropic effect of isoproterenol (0.01 μ m). In parallel studies carried out in rabbit AV nodal myocytes, intracellular mechanisms whereby NO mediates the inhibitory effect of adenosine on isoproterenol-induced A–H interval shortening were studied. Adenosine (3 μ m) inhibited isoproterenol-stimulated (0.1 μ m) ICa,L(β -ICa,L) by 46±6% (P<0.001, n=17). Consistent with isolated heart data, the NOS inhibitors, l -NMMA (100 μ m) and L-NNA (500 μ m) attenuated the effect of adenosine onβ -ICa,Lby 69±8% (P<0.001, n=16) and 69±7% (P<0.001, n=10), respectively. An inhibitor of NO-stimulated guanylyl cyclase LY83538 (40 μ m) reduced the inhibitory effect of adenosine on β -ICa,Lby 97±6% (P=0.004,n =15). Similarly, the non-specific inhibitor of cAMP-phosphodiesterases IBMX (50 μ m) decreased the anti-adrenergic effect of adenosine by 60% (P=0.02, n=6), whereas the extracellular application of the non-hydrolyzeable cAMP analog 8-Br-cAMP (500 μ m) prevented this action of adenosine. Activation of cGMP-dependent protein kinase (PKG) by CPT-cGMP (300 μ m) diminished β -ICa,L, but to a significantly smaller degree (16±4%, P=0.025, n=12) than that caused by adenosine. NO mediates the anti-adrenergic effect of adenosine on AV nodal conduction by a mechanism predominately involving activation of cGMP-dependent cAMP-phosphodiesterase and to a lesser extent activation of PKG.

An investigation of the role played by the E-4031-sensitive (rapid delayed rectifier) potassium current in isolated rabbit atrioventricular nodal and ventricular myocytes.

The aim of this study was to measure and compare the profile of rapid delayed rectifier potassium current (IKr) elicited by action potential (AP) waveforms applied to isolated rabbit atrioventricular nodal (AVN) and ventricular myocytes. All measurements were made using whole-cell patch-clamp recordings at 37 degrees C. In AVN myocytes, IKr during voltage steps and slow ramp depolarisations showed "inward rectification" (characteristic for this channel) at positive potentials. The E-4031-sensitive current showed half-maximal activation at -10.8 +/- 0.86 mV, with a slope factor for the activation relation of 6.5 +/- 0.77 mV (n = 7). During AVN APs, IKr rapidly reached a peak after the AP upstroke and remained at similar amplitude until late in AP repolarisation. At the maximum diastolic potential following the AVN AP, a component of IKr remained which decayed during the pacemaker depolarisation, consistent with a role for the current in generating AVN pacemaker activity. In ventricular myocytes IKr was small at the beginning of the AP, and increased slowly during the AP plateau. Measurement of Ba-sensitive-inward rectifier K current (IK1) in ventricular myocytes revealed that IK1 rapidly increased during the final AP repolarisation phase, whilst IKr declined. It is concluded that IKr may participate in both AP repolarisation and the pacemaker depolarisation in AVN cells, whilst in ventricular myocytes, IKr and IK1 participate in controlling early and final AP repolarisation respectively.

Characteristics of a transient outward current (sensitive to 4-aminopyridine) in Ca2+-tolerant myocytes isolated from the rabbit atrioventricular node.

A transient outward current (Ito) has been observed in the atrioventricular node (AVN), but its characteristics in Ca-tolerant AVN myocytes have not been investigated previously. In this study, Ito was measured from Ca-tolerant rabbit AVN myocytes at 37 degrees C, using the whole-cell patch-clamp technique. With interfering currents inhibited, 500-ms voltage-clamp pulses applied from -80 mV elicited Ito at potentials positive to -30 mV, which increased in magnitude with test potential amplitude. This current was completely blocked by external application of 5 mM 4-aminopyridine (4-AP). During a command pulse, Ito activated rapidly then inactivated with a bi-exponential time-course. Fast and slow time constants of current inactivation (tauf and taus, respectively) showed voltage dependence. At 0 mV, tauf was 14.5+/-2.7 ms and taus was 112.8+/-21. 2 ms, whilst at +60 mV tauf was 6.7+/-1.1 ms and taus was 63.7+/-9.2 ms (n=25). The steady-state inactivation relationship showed half-maximal inactivation at -33.8 mV (n=8). Re-activation of Ito after an inactivating pre-pulse showed a bi-exponential time-course of recovery: tau1 was 196+/-70 ms, and tau2 was 2707+/-1010 ms (n=6, at -80 mV). Repetitive application of voltage-clamp test pulses showed that Ito inactivation accumulated on repetitive stimulation, but reached a steady state rapidly for a given pulse frequency (0. 2-1.0 Hz). AVN Ito was sensitive to the class 1 anti-arrhythmic flecainide (EC50 for peak current of 24 microM), which showed selectivity for the rapidly inactivating current component. Quinidine also inhibited Ito in a dose-dependent fashion, but did not affect the current time-course. Under voltage-clamp conditions, a simulated diastolic depolarisation from -70 to -45 mV did not significantly reduce Ito amplitude, and under current-clamp conditions 4-AP inhibited spontaneous action potentials. Although this is consistent with a significant role for Ito in shaping AVN activity, under the conditions of this study 4-AP also partially blocked the "rapid" delayed rectifier current, IKr, and so the effects of 4-AP on action potentials could not be attributed exclusively to its effects on Ito.

Hyperkalemia enhances the effect of adenosine on IK,ADO in rabbit isolated AV nodal myocytes and on AV nodal conduction in guinea pig isolated heart.

The atrioventricular (AV) node is insensitive to changes in extracellular potassium concentration, [K+]o, because of the absence of the inward rectifier potassium current (IK1). However, we propose that in the presence of adenosine, elevated [K+]o should increase the adenosine-activated inward rectifier potassium current (IK,ADO) in AV nodal myocytes and hence augment the negative dromotropic effect of the nucleoside. The effects of normal (4.8 mmol/L) and high (8.0 mmol/L) [K+]o on adenosine-induced changes in resting membrane potential (Vm), IK,ADO, and membrane resistance (Rm) in rabbit isolated AV nodal myocytes and in AV nodal conduction delay (atrium-to-His bundle, AH, interval) in guinea pig isolated hearts were determined with the use of whole-cell patch-clamp and His bundle electrogram techniques, respectively. High [K+]o alone did not significantly affect membrane current, Rm, or Vm in AV nodal myocytes. However, high [K+]o in the presence of adenosine (3 micromol/L) markedly increased Im (-0. 249+/-0.038 to -0.571+/-0.111 nA, P<0.05) at -100 mV and reduced Rm (151+/-21 to 77+/-8 MOmega, P<0.02). Adenosine still hyperpolarized Vm from -48+/-2 to -65+/-1 mV (P<0.001). High [K+]o alone did not significantly affect the AH interval in isolated hearts. However, high [K+]o markedly lengthened the AH interval prolongation caused by adenosine (4 micromol/L, 7.9+/-0.8 vs 22.1+/-3.0 ms, P<0.001). The potentiating effect of high [K+]o on adenosine-induced delay in AV nodal conduction was abolished by BaCl2 (100 micromol/L). By increasing IK,ADO and decreasing Rm of AV nodal myocytes, elevated [K+]o, augments the depressant effect of adenosine on AV nodal conduction.

Modulation by mexiletine of action potentials, L-type Ca current and delayed rectifier K current recorded from isolated rabbit atrioventricular nodal myocytes.

Using whole-cell patch clamp recordings at 37 degrees C, we have examined the effects of externally applied mexiletine (a class 1b antiarrhythmic agent) on action potentials, L-type Ca current (ICa, L) and delayed rectifier K current (IK) in single isolated rabbit atrioventricular nodal (AVN) myocytes. In spontaneously active AVN cells, 30-100 micro;M mexiletine depolarised the maximum diastolic potential and slowed both action potential upstroke and repolarisation. Under selective recording conditions for ICa,L, mexiletine reduced peak ICa,L (at +10 mV) amplitude in a dose-dependent fashion (41.8 +/- 3.0% inhibition by 100 micro;M and 16.4 +/- 1.8% at 30 micro;M). The voltage dependence of ICa,L activation was unaffected by both concentrations of the drug. Under selective recording conditions, IK amplitude was measured as the peak of the deactivating tail current following a depolarising voltage pulse to +20 mV. 30 micro;M mexiletine inhibited IK by 34.3 +/- 5.8%, whilst 100 micro;M mexiletine reduced the current by 52.7 +/- 6.1%. The effects of mexiletine on ICa,L and IK are likely to contribute significantly to the changes in action potentials observed in spontaneously active cells. These findings are also suggestive of key roles for ICa,L and IK in determining the shape and rate of action potentials in this region of the heart.

Electrotonic modulation of electrical activity in rabbit atrioventricular node myocytes.

Electrotonic effects of electrically coupling atrioventricular (AV) nodal cells to each other and to real and passive models of atrial and ventricular cells were studied using a technique that does not require functional gap junctions. Membrane potential was measured in each cell using suction pipettes. Mutual entrainment of two spontaneously firing AV nodal cells was achieved with a junctional resistance (Rj) of 500 M omega, which corresponds to only 39 junctional channels, assuming a single-channel conductance of 50 pS. Coupling of AV nodal and atrial cells at Rj of 50 M omega caused hyperpolarization of the nodal cell, decreasing its action potential duration and either slowing or blocking diastolic depolarization in the AV node myocyte. Opposite changes occurred in the atrial action potential. When AV nodal and ventricular cells were coupled at Rj of 50 M omega, nodal diastolic potential was markedly hyperpolarized and diastolic depolarization was completely blocked with little change in ventricular diastolic potential. However, coupling did elicit marked changes in the action potential duration of both cells, with prolongation in the nodal cell and shortening in the ventricular cell. Nodal maximum upstroke velocity was increased by both atrial and ventricular coupling, as expected from the hyperpolarization that occurred. With an Rj of 50 M omega, spontaneous firing was blocked in all single AV nodal pacemaker cells during coupling to a real or passive model of an atrial or ventricular cell. These results demonstrate that action potential formation and waveform in a single AV nodal cell is significantly affected by electrical coupling to other myocytes.

Role of nitric oxide, cyclic GMP and superoxide in inhibition by adenosine of calcium current in rabbit atrioventricular nodal cells.

To study the intracellular pathways which mediate the inhibitory actions of adenosine on isoprenaline-stimulated calcium current (ICa) in atrioventricular (AV) nodal myocytes. The whole-cell patch-clamp technique was used to record ICa from rabbit AV nodal cells, isolated by enzymatic and mechanical dispersion. Isoprenaline, 0.1 microM, increased peak ICa from 0.58 +/- to 1.23 +/- 0.1 nA, and this increase was reversibly inhibited by adenosine, 10 microM (83 +/- 6%), which we have previously shown to be mediated by nitric oxide (NO) production. A membrane-permeable analogue of cyclic GMP, 8-Br-cGMP (300 microM), an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect of adenosine on ICa-Methylene blue (10 microM), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide (.02-), did not prevent, but increased, the inhibiting action of adenosine (49.5 +/- 6.6%, P < 0.01). Methylene blue (50 microM) caused a reduction of ICa, with further inhibition when combined with adenosine. A .O(2-)-generating system, xanthine oxidase (0.02 U/ml) and purine (2.3 mM), also increased the inhibitory action of adenosine on ICa. Inhibition of ICa by adenosine in the presence of xanthine oxidase was not prevented by 8-Br-cGMP (300 microM) and was not influenced by pre-incubation of cells with a NO synthase inhibitor, L-NAME (0.5 mM). The inhibitory effect of adenosine on ICa in rabbit AV nodal myocytes can be mediated by two mechanisms--stimulation of cGMP-stimulated phosphodiesterase by NO-induced cGMP, and a mechanism which involves interaction with .O2- production.

Inhibition of L-type calcium current by propafenone in single myocytes isolated from the rabbit atrioventricular node.

1. The atrioventricular node (AVN) is an important part of the conduction system in the heart and is a significant site of antiarrhythmic drug action. The class 1 antiarrhythmic propafenone is effective in treating a variety of arrhythmias, including those involving the AVN. In this study, we have investigated the effects of propafenone on ionic currents in single rabbit AVN cells, focusing in particular on those on L-type calcium current (ICa,L). 2. With a standard K-based internal dialysis solution, exposure to 5 microM propafenone reduced significantly the amplitude of ICa,L. In spontaneously active AVN myocytes, action potential upstroke velocity was decreased by propafenone exposure, consistent with the observed change in ICa,L. 3. By use of a Cs-based internal dialysis solution to record ICa,L selectively, voltage clamp test pulses were applied from a holding potential of -40 mV to +10 mV (stimulation frequency 0.33 Hz). Propafenone 5 microM reduced mean ICa,L density at +10 mV from -9.58 +/- 1.05 pA/pF to -4.19 +/- 0.60 pA/pF (P < 0.002). A range of propafenone concentrations were applied which reduced ICa,L in a dose-dependent manner (IC50 1.7 microM). When test pulses were applied to a range of potentials, propafenone reduced ICa,L at each potential without significantly affecting the activation curve for this current. Thus, propafenone reduced ICa,L conductance, without affecting the voltage-dependent activation properties of the current. 4. ICa,L block by propafenone exhibited tonic-, use- and frequency-dependent characteristics. 5. In the presence of propafenone, the voltage-dependence of inactivation of ICa,L was shifted 8 mV in the hyperpolarizing direction. Also, the recovery of ICa,L from inactivation was slowed by propafenone. 6. The ICa,L blocking properties of propafenone may mediate some of the antiarrhythmic properties of this agent, particularly in regions of the heart such as the AVN in which ICa,L contributes significantly to the action potential upstroke.

The actions of nickel on membrane currents activated by hyperpolarisation in single cells from the rabbit atrioventricular node

1. 1. The atrioventricular node (AVN) is vital for cardiac function as it normally provides the only conduction route for the cardiac impulse from atria to ventricles and can act as a pacemaker for the ventricles if the sinoatrial node (SAN) fails. We have shown previously that whilst 80–90% of AVN myocytes do not possess I f (we have termed these type I cells), a small proportion (10–20%) of AVN cells (type 2) do exhibit I f. 2. 2. The present study describes the effects of the divalent cation nickel (Ni) on membrane currents activated by hyperpolarising voltage clamps from −40/mV in type 1 and type 2 cells at 35°C, using the whole cell patch clamp technique. In type 2 cells 5 mM Ni enhanced the amplitude of I f. At −120 mV the mean Ni-activated I f was −1.85 ± 0.28 pA/pF (mean ± SEM; n = 5). Ni significantly enhanced I f at −70 mV and at all potentials negative to this ( p < 0.05 at −70, −80, −90 and −110 mV; 0.05 < p < 0.1 at −100 mV; p < 0.005 at −120 mV). 3. 3. In type 1 cells, which exhibit a small time-independent inward current on hyperpolarisation there was no activation of If by Ni ( p > 0.1 at all potentials between −40 mV and −120 mV). 4. 4. In type 1 cells 5 mM Ni significantly reduced the time-independent inward current activated by a hyperpolarising pulse to −120 mV ( p < 0.02) and had a smaller effect at −110 and −100 mV (0.05 < p < 0.1 at these potentials). With pulses to less negative potentials there was no significant alteration of the time-independent current. 5. 5. An additional observation was that the fast sodium current activated on repolarisation of the membrane potential to −40 mV after a hyperpolarising voltage clamp appeared to be blocked by Ni. However, this apparent blockade reflected a positive shift in the activation threshold for I Na, since a repolarising step to −30 mV could still elicit I Na. 6. 6. Ni is known to block sarcolemmal Na/Ca exchange in cardiac cells, and one possible mechanism for the enhancement of I f by Ni in type 2 cells is increased intracellular Ca via Na/Ca exchange blockade increasing I f. The reduction in end pulse current in type 1 cells is also consistent with Na/Ca exchange current blockade. A second possibility of the enhanced I f in type 2 cells with Ni is a positive shift of the activation curve for I f in the presence of an increased concentration of external divalent cations.

Adenosine increases potassium conductance in isolated rabbit atrioventricular nodal myocytes

Adenosine increases potassium conductance in isolated rabbit atrioventricular nodal myocytes

Adenosine increases potassium conductance in isolated rabbit atrioventricular nodal myocytes

To study the actions of adenosine on the electrophysiology of spontaneously active, rod-shaped cells enzymatically isolated from rabbit atrioventricular (AV) node. Calcium-tolerant myocytes were isolated from the region of the AV node by enzymatic and mechanical dispersion. They were rod- or spindle-shaped, with spontaneous activity at 35-37 degrees C, and had higher membrane resistances (776 +/- 283 M omega, n = 13), compared to atrial cells (41 +/- 18.2 M omega, n = 7; P < 0.001). Membrane potential, spontaneous action potentials and transmembrane ionic currents were studied using the whole-cell patch-clamp technique, in current-clamp and voltage-clamp mode. Adenosine (0.1-50 microM) slowed or abolished the spontaneous activity, with hyperpolarisation of the membrane potential. Voltage-clamp experiments showed that adenosine induced an inwardly rectifying time-independent current. The adenosine-induced current was shown to be carried by potassium ions by the effect of increasing external potassium, which altered the reversal potential in accordance with the calculated potassium equilibrium potential. The A1 adenosine receptor antagonist, CPDPX (8-cyclopentyl-1,3-dypropylxanthine), reversed the effects of adenosine and an A1 receptor agonist, R-PIA [R(-)N(6)-(2-phenylisopropyl)adenosine] had effects similar to adenosine. Adenosine also caused a small decrease in inward calcium current (ICa) in some AV nodal cells. These results indicate that adenosine acts at A1 adenosine receptors to suppress spontaneous activity, hyperpolarise membrane potential and induce a time-independent potassium current in AV nodal cells. These actions, combined with reduction in inward calcium current in some cells, may underlie the negative chronotropic and dromotropic actions of adenosine on rabbit AV nodal cells.

The hyperpolarisation-activated current,I f, is not required for pacemaking in single cells from the rabbit atrioventricular node

The atrioventricular node (AVN) is vital for cardiac function. One of its properties is that it can act as a pacemaker for the ventricles if the sinoatrial node fails. This study investigates the role of the hyperpolarisation-activated inward current (I(f)) in generating pacemaker activity in morphologically normal single cells isolated from the rabbit AVN. Whole-cell patch-clamp recordings show that 80%-90% of AVN myocytes do not possess I(f), but nevertheless generate spontaneous action potentials with normal pacemaker depolarisations before each action potential upstroke. We have termed this type of cell "type 1". A small proportion (10%-20%) of spontaneously active AVN cells (type 2) do exhibit I(f). A 100 nM solution of isoprenaline increased the action potential rate of type 1 cells by 31%. In these cells isoprenaline did not activate any I(f) whereas in type 2 cells it clearly increased the amplitude of I(f). Manganese at 2 mM also increased the amplitude of I(f) in type 2 cells, but did not reveal I(f) in type 1 cells. We conclude that, whilst I(f) may play a role in modulating pacemaker activity in type 2 cells, in the majority of AVN cells (type 1) pacemaker depolarisation normally occurs in the complete absence of I(f). Furthermore, the inability of both isoprenaline and Mn to reveal I(f) in type 1 cells suggests that I(f) channels may be absent in these cells.

Ionic mechanisms of electronic inhibition and concealed conduction in rabbit atrioventricular nodal myocytes.

The term "concealed conduction" is used in electrocardiography when a proximal (atrial or ventricular) impulse penetrates the atrioventricular (AV) node but fails to traverse it completely. Its penetration into the node is inferred by its after effects on the propagation of succeeding impulses. Concealed AV nodal conduction is a well-established phenomenon, but its precise cellular and subcellular mechanisms are unknown. It has been suggested that concealed conduction results from a transient impairment of excitability caused by the subthreshold depolarization (ie, electrotonic inhibition) that is elicited downstream of the site of block. We studied the ionic mechanism of electrotonic inhibition and concealed conduction in single myocytes isolated from the rabbit AV node. Cells were paced using just-threshold current pulses delivered at a constant (basic) cycle length of 1 second. Appropriately timed interpolation of a conditioning pulse of depolarizing but subthreshold current led to failure of the subsequent, previously successful activation. The ability of the subthreshold response to inhibit subsequent excitation was increased when the interval between the conditioning and succeeding pulses was shortened, when the amplitude of the conditioning pulse was increased, or when the inward sodium current was blocked by superfusion with tetrodotoxin (30 microM). Voltage clamp analysis demonstrated that electrotonic inhibition results from partial inactivation of the transient calcium current (ICa.T). Similar results were obtained using a mathematical model (Hodgkin-Huxley type) of the AV nodal myocyte. Additional simulations in a linear array of AV nodal cells showed that when a premature impulse fails to traverse the AV node, the subthreshold depolarization elicited downstream of the site of block may lead to a transient reduction of excitability with consequent delay or block of the succeeding impulse. The overall data strongly suggest that some of the electrocardiographic manifestations of concealed AV conduction are the result of electrotonic inhibition of excitability secondary to a transient decrease in ICa.T.

A method for isolating rabbit atrioventricular node myocytes which retain normal morphology and function

This report describes a method for isolating single rabbit atrioventricular (AV) node myocytes which retain their normal morphology when exposed to millimolar levels of calcium. Previous attempts to isolate cells from the AV node have produced myocytes that "round up" (i.e., go into contracture) when exposed to calcium. We show that the cells isolated with our technique possess properties similar to those described for intact AV nodal tissue. We find that single AV node myocytes are shorter and thinner (mean dimension = 103.5 +/- 2.3 by 7.8 +/- 0.2 microns; mean +/- SE, n = 90) than atrial or ventricular cells. Many of the cells produced by this isolation procedure generate spontaneous action potentials (188 +/- 9 beats/min; n = 6), which resemble action potentials recorded previously from the intact AV node. Voltage-clamp recordings from spontaneously active cells revealed similar membrane currents to those seen in intact tissue: fast sodium current and a L-type calcium current, followed by a delayed outward current. However, we found little evidence for the hyperpolarization-activated current (I(f)). Because the cells responded normally to concentrations of acetylcholine and isoproterenol within the physiological range, their cholinergic and adrenergic receptors appear to be well preserved by the isolation procedure. The ability to isolate morphologically and functionally normal AV myocytes may represent a significant advance for the investigation of nodal physiology at the cellular level.

Wenckebach periodicity in single atrioventricular nodal cells from the rabbit heart.

Previous studies have suggested that Wenckebach periodicity in cardiac tissues may occur because of discontinuous propagation across junctional areas in which there is high intercellular resistivity or different cell types. Under these conditions, the impulse may stop altogether at a given junction, or may renew its propagation but only after a step delay imposed by the diastolic time-dependent recovery in the excitability of cells distal to that junction. Accordingly, Wenckebach periodicity in the atrioventricular node may be explained in terms of electrotonically mediated delay in the activation of the nodal cells. To test this hypothesis, we have studied recovery of excitability, and susceptibility to rate-dependent activation failure in single myocytes isolated from the adult rabbit atrioventricular node. Recordings were obtained by using the patch technique in the whole-cell, current clamp configuration. Repetitive stimulation of single atrioventricular nodal myocytes with depolarizing current pulses of critical amplitude yielded frequency-dependent stimulus response patterns that ranged from 1:1, through various Wenckebachlike periodicities (e.g., 5:4 and 4:3) to 2:1 and 3:1. Both typical and atypical Wenckebach structures were demonstrated, as well as "complex" patterns (e.g., reverse Wenckebach or alternating Wenckebach) previously ascribed to multiple levels of block. The diastolic recovery of excitability curve, determined by application of repetitive stimuli at cycle lengths that were longer than the action potential duration, showed a monotonic function with a refractory period outlasting the action potential duration (i.e., postrepolarization refractoriness). Abbreviation of the stimulation cycle length to values below those of the action potential duration revealed the existence of a period of supernormal excitability during the repolarizing phase of the action potential. In either case, the stimulus response patterns obtained were a direct consequence of the shape of the recovery of excitability curve. The monotonic portion of the recovery curve was fitted to an empirical equation that when iterated reproduced the stimulus response patterns observed in the atrioventricular nodal cell. Our data demonstrate that recovery of excitability after an action potential is indeed a function of the diastolic interval, and that this slow process sets the conditions for the development of Wenckebach periodicity in the atrioventricular node.