A gradient model of cardiac pacemaker myocytes

We have formulated a spatial-gradient model of action potential heterogeneity within the rabbit sinoatrial node (SAN), based on cell-specific ionic models of electrical activity from its central and peripheral regions. The ionic models are derived from a generic cell model, incorporating five background and exchange currents, and seven time-dependent currents based on three- or four-state Markov schemes. State transition rates are given by non-linear sigmoid functions of membrane potential.

By appropriate selection of parameters, the generic model is able to accurately reproduce a wide range of action potential waveforms observed experimentally. Specifically, the model can fit recordings from central and peripheral regions of the SAN with RMS errors of 0.3987 and 0.7628 mV, respectively. Using a custom least squares parameter optimisation routine, we have constructed a spatially-varying gradient model that exhibits a smooth transition in action potential characteristics from the central to the peripheral region, whilst ensuring individual membrane currents remain physiologically accurate. Smooth transition action potential characteristics include maximum diastolic potential, overshoot potential, upstroke velocity, action potential duration and cycle length. The gradient model is suitable for developing higher dimensional models of the right atrium, in which action potential heterogeneity within nodal tissue may be readily incorporated.     

Role of pacemaking current in cardiac nodes: insights from a comparative study of sinoatrial node and atrioventricular node

Cardiac pacemaking in the sinoatrial (SA) node and atrioventricular (AV) node is generated by an interplay of many ionic currents, one of which is the funny pacemaker current (I_f). To understand the functional role of I_f in two different pacemakers, comparative studies of spontaneous activity and expression of the HCN channel in mouse SA node and AV node were performed. The intrinsic cycle length (CL) is 179±2.7 ms (n=5) in SA node and 258±18.7 ms (n=5) in AV node. Blocking of I_f current by 1 μmol/L ZD7288 increased the CL to 258±18.7 ms (n=5) and 447±92.4 ms (n=5) in SA node and AV node, respectively. However, the major HCN channel, HCN4 expressed at low level in the AV node compared to the SA node. To clarify the discrepancy between the functional importance of I_f and expression level of HCN4 channel, a SA node cell model was used. Increasing the I_f conductance resulted in decreasing in the CL in the model, which explains the high pacemaking rate and high expression of HCN channel in the SA node. Resistance to the blocking of I_f in the SA node might result from compensating effects from other currents (especially voltage sensitive currents) involved in pacemaking. The computer simulation shows that the difference in the intrinsic CL could explain the difference in response to I_f blocking in these two cardiac nodes.     

Bifurcation analysis and effects of changing ionic conductances on pacemaker rhythm in a sinoatrial node cell model

The electrical excitation (action potential generation) of sinoatrial node (cardiac pacemaker) cells is directly related to various ion channels (pore-forming proteins) in cell membranes. In order to analyze the relation between action potential generation and ion channels, we use the Yanagihara-Noma-Irisawa (YNI) model of sinoatrial node cells, which is described by the Hodgkin-Huxley-type equations with seven variables. In this paper, we analyze the global bifurcation structure of the YNI model by varying various conductances of ion channels, and examine the effects of these conductance changes on pacemaker rhythm (frequency of action potential generation). The coupling effect on pacemaker rhythm is also examined approximately by applying external current to the YNI model.     

Inhomogeneity in the response to mechanical stimulation: cardiac muscle function and gene expression

Mechanical stimulation has important consequences for myocardial function. However, this stimulation and the response to it, is not uniform. The right ventricle is thinner walled and operates at lower pressure than the left ventricle. Within the ventricles, differences in the orientation of myocardial fibres exist. These differences produce inhomogeneity in the stress and strain between and across the ventricles. Possibly as a result of these variations in mechanical stimulation, there are well characterised inhomogeneities in gene expression and protein function within the ventricular myocardium, for example in the transient outward K+ current and its associated Kv channels. Perhaps not surprisingly, it is becoming apparent that gradients of expression and function exist for proteins that are intimately involved in the response to mechanical stimulation in the heart, for example in the left ventricle of the rat there is a transmural gradient in mRNA and current density of the mechanosensitive two-pore domain K+ channel TREK-1 (ENDO>EPI). In healthy hearts it is assumed that these gradients are important for normal function and therefore that their disruption in diseased myocardium is involved in the dysfunction that occurs.     

Single chloride-permeable channels of large conductance in cultured cardiac cells of new-born rats

Large conductance channels were observed in the membrane of cultured cardiac cells of newborn rats studied with the patch-clamp technique in cell-attached and inside-out configurations. These channels were observed in 4% of the patches. In the cell-attached configuration they exhibited outward rectification and partial inactivation. In the inside-out configuration no rectification occurred but inactivation was present, mainly during hyperpolarizations. Two channels with large single unit conductances (400–450 pS) and one with a smaller conductance (200–250 pS) were frequently observed in the same patch. The two large channels generally had different kinetics. Under steady-state conditions the opening probability of the faster channel appeared to be voltage-independent. The slower channel was activated by depolarization. In asymmetrical solutions the permeability ratios P _Na/P _Cl were 0.03 and 0.24 for the larger and smaller channels, respectively; corresponding values for P _Ba/P _Cl were 0.04 and 0.09. It is proposed that in cardiac membranes the chloride permeability system is composed of widely dispersed microclusters forming grouped channels of different types and sizes.     

The role of connexin40 in developing atrial conduction

Connexin40 (Cx40) is the main connexin expressed in the murine atria and ventricular conduction system. We assess here the developmental role of Cx40 in atrial conduction of the mouse. Cx40 deficiency significantly prolonged activation times in embryonic day 10.5, 12.5 and 14.5 atria during spontaneous activation; the severity decreased with increasing age. In a majority of Cx40 deficient mice the impulse originated from an ectopic focus in the right atrial appendage; in such a case the activation time was even longer due to prolonged activation. Cx40 has thus an important physiological role in the developing atria.     

Mechanisms of up-regulation of single calcium channels by serotonin in Helix pomatia neurons

Action of serotonin (5-HT) on single Ca(2+) channel activity was studied in identified neurons of snail Helix pomatia. Only one type of Ca(2+) channels of 5 pS unitary conductance was determined under patch-clamp cell-attached mode. Kinetic analysis have shown a monotonically declining distribution of channel open times (OT) with mean time constant of 0.2 ms. The distribution of channel closed times (CT) could be fitted by double-exponential curve with time constants 1 and 12 ms. We established that 5-HT acts on Ca(2+) channel activity indirectly via cytoplasm. 5-HT prolonged the OT (up to 0.3 ms) and shortened the CT proportionally for both constants to 0.4 and 6 ms correspondingly. A conclusion is made that enhancement of Ca(2+) macro-current by 5-HT is determined by kinetic changes, increase of the number of active channels, and increase of the probability of OT. At the same time the transmitter did not affect the unitary channel conductance.     

Some aspects of the physiological role of ion channels in the nervous system

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.     

In-vivo phosphorylation of the cardiac L-type calcium channel beta-subunit in response to catecholamines

In canine myocardium, the -subunit of the L-type Ca²⁺ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217–222, 1993). We have assessed the identity of the -subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca²⁺ channel -subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the -subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dt_max. The results imply the involvement of a PKA-mediated phosphorylation of the Ca²⁺ channel -subunit in the adrenergic stimulation of intact canine myocardium.     

Difference equation model of ventricular parasystole as an interaction between cardiac pacemakers based on the phase response curve

A model of extended ventricular parasystole proposed by Moe et al. (1977) was formulated as a system of nonlinear difference equations by using the phase response curve of myocardial pacemakers. A number of ECG patterns of ventricular arrhythmia such as bigeminy, trigeminy etc. were explained from the property of periodic solutions of the equation. Characteristic properties of special kinds of arrhythmia called “concealed bigeminy” and “concealed trigeminy” were derived mathematically by assuming the model, in relation to the equation of the analog neuron model. The present study was considered to be of clinical significance as a theoretical foundation for the study of genesis of cardiac arrhythmias.     

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).     

Closure of ionic channels in turtle cones

Recent observations from turtle cones on the kinetics of the light response and the variance and power spectrum of spontaneous noise are discussed in terms of the behaviour of membrane ionic channels.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Overdrive excitation in the guinea pig sinoatrial node superfused in high [K+]o

The aim of the present experiments was to study the characteristics and mechanisms of the rhythm induced by overdrive (overdrive excitation, ODE) in the sinoatrial node (SAN) superfused in high [K⁺]_o (8–14 mM). It was found that: (1) overdrive may induce excitation in quiescent SAN and during a slow drive; (2) in spontaneously active SAN, overdrive may accelerate the spontaneous discharge; (3) immediately after the end of overdrive, a pause generally precedes the onset of the induced rhythm; (4) during the pause, an oscillatory potential (V_os) may be superimposed on the early diastolic depolarization (DD); (5) during the subsequent late DD, a different kind of oscillatory potential appears near the threshold for the upstroke (ThV_os) which is responsible for the initiation of spontaneous activity; (6) once started, the induced rhythm is fastest soon after overdrive; (7) faster drives induce longer and faster spontaneous rhythms; (8) the induced action potentials are slow responses followed by DD with a superimposed V_os, but ThV_os is responsible for ODE; (9) the induced rhythm subsides when ThV_os miss the threshold and gradually decay; (10) low [Ca²⁺]_o abolishes ODE; (11) in quiescent SAN, high [Ca²⁺]_o induces spontaneous discharge through ThV_os and increases its rate by enhancing V_os and shifting the threshold to more negative values, and (12) tetrodotoxin abolishes ODE as well as the spontaneous discharge induced by high [Ca²⁺]_o. In conclusion, in K⁺-depolarized SAN, ODE may be present in the apparent absence of calcium overload, is Ca²⁺- and Na⁺-dependent and is mediated by ThV_os and not by V_os.     

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.     

Magnetically induced electric fields and currents in the circulatory system

Blood flow in an applied magnetic field gives rise to induced voltages in the aorta and other major arteries of the central circulatory system that can be observed as superimposed electrical signals in the electrocardiogram (ECG). The largest magnetically induced voltage occurs during pulsatile blood flow into the aorta, and results in an increased signal at the location of the T-wave in the ECG. Studies involving the measurement of blood pressure, blood flow rate, heart sounds, and cardiac valve displacements have been conducted with monkeys and dogs exposed to static fields up to 1.5 tesla (T) under conditions producing maximum induced voltages in the aorta. Results of these studies gave no indication of alterations in cardiac functions or hemodynamic parameters. Cardiac activity monitored by ECG biotelemetry during continuous exposure of rats to a 1.5-T field for 10 days gave no evidence for any significant changes relative to the 10 days prior to and following exposure. Theoretical modeling of magnetic field interactions with blood flow has included a complete solution of the equation describing the flow of an electrically conductive fluid in the presence of a magnetic field (the Navier–Stokes equation) using the finite element technique. Magnetically induced voltages and current densities as a function of the applied magnetic field strength have been calculated for the aorta and surrounding tissues structures, including the sinoatrial node. Induced current densities in the region of the sinoatrial node are predicted to be >100 mA/m² at field levels >5 T in an adult human under conditions of maximum electrodynamic coupling with aortic blood flow. Magnetohydrodynamic interactions are predicted to reduce the volume flow rate of blood in the human aorta by a maximum of 1.3%, 4.9%, and 10.4% at field levels of 5, 10, and 15 T, respectively.     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs—fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca²⁺ permeable ion channel using Ca²⁺ indicators like fluo-3. These Single Channel Ca²⁺ Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca²⁺ sparks and Ca²⁺ puffs caused by Ca²⁺ release from intracellular stores (due to the opening of ryanodine receptors and IP₃ receptors, respectively). In contrast to intracellular Ca²⁺ release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca²⁺ handling in the vicinity of a channel with a known Ca²⁺ influx, to obtain the Ca²⁺ current passing through plasma membrane cation channels in near physiological solutions, to localize Ca²⁺ permeable ion channels on the plasma membrane, and to estimate the Ca²⁺ currents underlying those elementary events where the Ca²⁺ currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca²⁺ channels, and stretch-activated channels. For the L-type Ca²⁺ channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca²⁺ currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

Distinct modes of blockade in cardiac ATP-sensitive K+ channels suggest multiple targets for inhibitory drug molecules

Elementary K⁺ currents were recorded at 19°C in inside-out patches from cultured neonatal rat cardiocytes to elucidate the block phenomenology in cardiac ATP-sensitive K⁺ channels when inhibitory drug molecules, such as the sulfonylurea glibenclamide, the phenylalkylamine verapamil or sulfonamide derivatives (HE 93 and sotalol), are interacting in an attempt to stress the hypothesis of multiple channel-associated drug targets.Similar to their adult relatives, neonatal cardiac K_(ATP) channels are characterized by very individual open state kinetics, even in cytoplasmically well-controlled, cell-free conditions; at –7 mV, _open(1) ranged from 0.7 to 4.9 msec in more than 200 patches and _open(2) from 10 to 64 msec—an argument for a heterogeneous channel population. Nevertheless, a common response to drugs was observed. Glibenclamide and the other inhibitory molecules caused long-lasting interruptions of channel activity, after cytoplasmic application, as if drug occupancy trapped cardiac K_(ATP) channels in a very stable, nonconducting configuration. The resultant NP₀ depression was strongest with glibenclamide (apparent IC₅₀ 13 nmol/liter) and much weaker with verapamil (apparent IC₅₀ 9 mol/liter), HE 93 (apparent IC₅₀ 29 mol/liter) and sotalol (apparent IC₅₀ 43 mol/ liter) and may have resulted from the occupancy of a single site with drug-specific affinity or of two sites, the high affinity glibenclamide target and a distinct nonglibenclamide, low affinity target.Changes in open state kinetics, particularly in the transition between the O₁ state and the O₂ state, are other manifestations of drug occupancy of the channel. Any inhibitory drug molecule reduced the likelihood of attaining the O₂ state, consistent with a critical reduction of the forward rate constant governing the O₁-O₁ transition. But only HE 93 (10 mol/liter) associated (with an apparent association rate constant of 2.3 × 10⁶ mol^–1 sec^–1) to shorten significantly _open(2) to 60.6 ± 6% of the predrug value, not the expected result when the entrance in and the exit from the O₂ state would be drug-unspecifically nfluenced. Sotalol found yet another and definitely distinctly located binding site to interfere with K⁺ permeation; both enantiomers associated with a rate close to 5×10⁵ mol^–1 sec^–1 with the open pore thereby flicker-blocking cardiac K_(ATP) channels. Clearly, these channels accommodate more than one drug-binding domain.