Virtual electrodes in cardiac tissue: a common mechanism for anodal and cathodal stimulation.

Traditional cable analyses cannot explain complex patterns of excitation in cardiac tissue with unipolar, extracellular anodal, or cathodal stimuli. Epifluorescence imaging of the transmembrane potential during and after stimulation of both refractory and excitable tissue shows distinctive regions of simultaneous depolarization and hyperpolarization during stimulation that act as virtual cathodes and anodes. The results confirm bidomain model predictions that the onset (make) of a stimulus induces propagation from the virtual cathode, whereas stimulus termination (break) induces it from the virtual anode. In make stimulation, the virtual anode can delay activation of the underlying tissue, whereas in break stimulation this occurs under the virtual cathode. Thus make and break stimulations in cardiac tissue have a common mechanism that is the result of differences in the electrical anisotropy of the intracellular and extracellular spaces and provides clear proof of the validity of the bidomain model.     

Examination of stimulation mechanism and strength-interval curve in cardiac tissue

Understanding the basic mechanisms of excitability through the cardiac cycle is critical to both the development of new implantable cardiac stimulators and improvement of the pacing protocol. Although numerous works have examined excitability in different phases of the cardiac cycle, no systematic experimental research has been conducted to elucidate the correlation among the virtual electrode polarization pattern, stimulation mechanism, and excitability under unipolar cathodal and anodal stimulation. We used a high-resolution imaging system to study the spatial and temporal stimulation patterns in 20 Langendorff-perfused rabbit hearts. The potential-sensitive dye di-4-ANEPPS was utilized to record the electrical activity using epifluorescence. We delivered S1-S2 unipolar point stimuli with durations of 2-20 ms. The anodal S-I curves displayed a more complex shape in comparison with the cathodal curves. The descent from refractoriness for anodal stimulation was extremely steep, and a local minimum was clearly observed. The subsequent ascending limb had either a dome-shaped maximum or was flattened, appearing as a plateau. The cathodal S-I curves were smoother, closer to a hyperbolic shape. The transition of the stimulation mechanism from break to make always coincided with the final descending phase of both anodal and cathodal S-I curves. The transition is attributed to the bidomain properties of cardiac tissue. The effective refractory period was longer when negative stimuli were delivered than for positive stimulation. Our spatial and temporal analyses of the stimulation patterns near refractoriness show always an excitation mechanism mediated by damped wave propagation after S2 termination.     

Effect of Global Cardiac Ischemia on Human Ventricular Fibrillation: Insights from a Multi-scale Mechanistic Model of the Human Heart

Acute regional ischemia in the heart can lead to cardiac arrhythmias such as ventricular fibrillation (VF), which in turn compromise cardiac output and result in secondary global cardiac ischemia. The secondary ischemia may influence the underlying arrhythmia mechanism. A recent clinical study documents the effect of global cardiac ischaemia on the mechanisms of VF. During 150 seconds of global ischemia the dominant frequency of activation decreased, while after reperfusion it increased rapidly. At the same time the complexity of epicardial excitation, measured as the number of epicardical phase singularity points, remained approximately constant during ischemia. Here we perform numerical studies based on these clinical data and propose explanations for the observed dynamics of the period and complexity of activation patterns. In particular, we study the effects on ischemia in pseudo-1D and 2D cardiac tissue models as well as in an anatomically accurate model of human heart ventricles. We demonstrate that the fall of dominant frequency in VF during secondary ischemia can be explained by an increase in extracellular potassium, while the increase during reperfusion is consistent with washout of potassium and continued activation of the ATP-dependent potassium channels. We also suggest that memory effects are responsible for the observed complexity dynamics. In addition, we present unpublished clinical results of individual patient recordings and propose a way of estimating extracellular potassium and activation of ATP-dependent potassium channels from these measurements.     

Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution

The heterogeneities of electrophysiological properties of cardiac tissue are the main factors that control both arrhythmia induction and maintenance. Although the local increase of extracellular potassium ([K(+)](o)) due to coronary occlusion is a well-established metabolic response to acute ischemia, the role of local [K(+)](o) heterogeneity in phase 1a arrhythmias has yet to be determined. In this work, we created local [K(+)](o) heterogeneity and investigated its role in fast pacing response and arrhythmia induction. The left marginal vein of a Langendorff-perfused rabbit heart was cannulated and perfused separately with solutions containing 4, 6, 8, 10, and 12 mM of K(+). The fluorescence dye was utilized to map the voltage distribution. We tested stimulation rates, starting from 400 ms down to 120 ms, with steps of 5-50 ms. We found that local [K(+)](o) heterogeneity causes action potential (AP) alternans, 2:1 conduction block, and wave breaks. The effect of [K(+)](o) heterogeneity on electrical stability and vulnerability to arrhythmia induction was largest during regional perfusion with 10 mM of K(+). We detected three concurrent dynamics: normally propagating activation when excitation waves spread over tissue perfused with normal K(+), alternating 2:2 rhythm near the border of [K(+)](o) heterogeneity, and 2:1 aperiodicity when propagation was within the high [K(+)](o) area. [K(+)](o) elevation changed the AP duration (APD) restitution and shifted the restitution curve toward longer diastolic intervals and shorter APD. We conclude that spatial heterogeneity of the APD restitution, created with regional elevation of [K(+)](o), can lead to AP instability, 2:1 block, and reentry induction.     

Mechanism of anode break stimulation in the heart.

Anodal stimulation is routinely observed in cardiac tissue, but only recently has a mechanism been proposed. The bidomain cardiac tissue model proposes that virtual cathodes induced at sites distant from the electrode initiate the depolarization. In contrast, none of the existing cardiac action potential models (Luo-Rudy phase I and II, or Oxsoft) predict anodal stimulation at the single-cell level. To determine whether anodal stimulation has a cellular basis, we measured membrane potential and membrane current in mammalian ventricular myocytes by using whole-cell patch clamp. Anode break responses can be readily elicited in single ventricular cells. The basis of this anodal stimulation in single cells is recruitment of the hyperpolarization-activated inward current I(f). The threshold of activation for I(f) is -80 mV in rat cells and -120 mV in guinea pig or canine cells. Persistent I(f) "tail" current upon release of the hyperpolarization drives the transmembrane potential toward the threshold of sodium channels, initiating an action potential. Time-dependent block of the inward rectifier, I(K1), at hyperpolarized potentials decreases membrane conductance and thereby potentiates the ability of I(f) to depolarize the cell on the break of an anodal pulse. Inclusion of I(f), as well as the block and unblock kinetics of I(K1), in the existing Luo-Rudy action potential model faithfully reproduces anode break stimulation. Thus active cellular properties suffice to explain anode break stimulation in cardiac tissue.     

Spatial distribution of cardiac transmembrane potentials around an extracellular electrode: dependence on fiber orientation.

Recent theoretical models of cardiac electrical stimulation or defibrillation predict a complex spatial pattern of transmembrane potential (Vm) around a stimulating electrode, resulting from the formation of virtual electrodes of reversed polarity. The pattern of membrane polarization has been attributed to the anisotropic structure of the tissue. To verify such model predictions experimentally, an optical technique using a fluorescent voltage-sensitive dye was used to map the spatial distribution of Vm around a 150-microns-radius extracellular unipolar electrode. An S1-S2 stimulation protocol was used, and vm was measured during an S2 pulse having an intensity equal to 10x the cathodal diastolic threshold of excitation. The recordings were obtained on the endocardial surface of bullfrog atrium in directions parallel and perpendicular to the cardiac fibers. In the longitudinal fiber direction, the membrane depolarized for cathodal pulses (and hyperpolarized for anodal pulses) but only in a region within 445 +/- 112 microns (and 616 +/- 78 microns for anodal pulses) from the center of the electrode (n = 9). Outside this region, vm reversed polarity and reached a local maximum at 922 +/- 136 microns (and 988 +/- 117 microns for anodal pulses) (n = 9). Beyond this point vm decayed to zero over a distance of 1.5-2 mm. In the transverse fiber direction, the membrane depolarized for cathodal pulses (and hyperpolarized for anodal pulses) at all distances from the electrode. The amplitude of the response decreased with distance from the electrode with an exponential decay constant of 343 +/- 110 microns for cathodal pulses and 253 +/- 91 microns for anodal pulses (n = 7). The results were qualitatively similar in both fiber directions when the atrium was bathed in a solution containing ionic channel blockers. A two-dimensional computer model was formulated for the case of highly anisotropic cardiac tissue and qualitatively accounts for nearly all the observed spatial and temporal behavior of vm in the two fiber directions. The relationships between vm and both the "activating function" and extracellular potential gradient are discussed.     

Steady potential and extracellular potassium shifts in the rat neocortex during sustained low-frequency electrical stimulation of the cortical surface

Cyclic excitation arising in the rat neocortex under the influence of low-frequency electrical stimulation was studied. The duration of the excitation cycles and intervals between them depended on the stimulation parameters. In the period of excitation the negative shift of steady potential reached 4–5 mV, and the extracellular potassium ion concentration, measured with the aid of potassium-selective microelectrodes, rose to 8–10 mM. The process of periodic excitation was not self-maintained and it ceased after the current was stopped. As a result of small doses of pentobarbital (10–20 mg/kg) the response thresholds rose, and intervals between excitation phases were increased by several times. Pentobarbital in a dose of 30–40 mg/kg completely abolished cyclic excitation for 2–3 h. Correlation between shifts of steady potential and extracellular potassium concentration differed in anesthetized and unanesthetized animals.     

Effect of sodium,potassium, and calcium ions on electroreceptor function in catfish

Impulses from single electroreceptors (small pit organs) of catfish (Ictalurus nebulosus) were recorded during stimulation by square pulses. Solutions with different concentrations of potassium, sodium, and calcium ions were applied to the pore of the receptor. Solutions with a low CaCl₂ concentration did not alter the responses of the receptor. Calcium ions in concentrations of over 5 mM increased the threshold of the response to electrical stimulation. The threshold to anodal stimulation was increased in solutions of 2 mM sodium and potassium and no response was given to a cathodal stimulus. The effect of 2 mM solutions of NaCl and KCl was abolished by the addition of 0.4 mM CaCl₂ or by application of a long anodal stimulus of high intensity (10⁻⁸∓10⁻⁷ A/mm²). Increasing the potassium ion concentration to 10–20 mM restored normal receptor function but a further increase led to elevation of the threshold. The action of an electric current is compared with the action of the ions.     

Potassium- and ionophore A23187-induced discharge of secretory protein in guinea pig pancreatic lobules. Role of extracellular calcium

Elevated concentrations of potassium chloride (50 to 120 mM) in the incubation medium stimulated in vitro discharge of secretory protein from guinea pig pancreatic lobules. The effect of potassium was not inhibited by 10(-4) M atropine, sodium substitutes, or 10(-5) M tetrodotoxin. Exposure of lobules to elevated concentrations of potassium chloride did not increase the release of tissue lactic dehydrogenase and resulted in the appearance of exocytotic images detected by electron microscopy. The time course and extent of discharge due to 75 mM KCl were similar to those caused by the ionophore A23187 and the secretory effect of both agents depended on extracellular calcium and intracellular energy reserves. Potassium chloride stimulation of 75 mM increased the influx of extracellular calcium by 49%, as measured by net 45Ca uptake. Optimal carbamylcholine chloride or pancreozymin stimulation consistently showed a greater effect on discharge than optimal KCl or A23187 stimulation and the additional effect depended on the ability of these physiological secretagogues to recruit calcium from intracellular sources. Potassium chloride stimulation did not result in cyclic GMP elevations in the presence of atropine and those elevations due to A23187 stimulation were small (21 to 30%) and dissimilar both in character (calcium dependence) and time course compared to those resulting from the physiological secretagogues. These findings allow us to define two interrelated pathways which couple hormonal stimulation and discharge of secretory protein in the exocrine pancreas.     

Taste transduction mechanism: similar effects of various modifications of gustatory receptors on neural responses to chemical and electrical stimulation in the frog

Responses in the frog glossopharyngeal nerve induced by electrical stimulation of the tongue were compared with those induced by chemical stimuli under various conditions. (a) Anodal stimulation induced much larger responses than cathodal stimulation, and anodal stimulation of the tongue adapted to 5 mM MgCl2 produced much larger responses than stimulation with the tongue adapted to 10 mM NaCl at equal current intensities, as chemical stimulation with MgCl2 produced much larger responses than stimulation with NaCl at equal concentration. (b) The enhansive and suppressive effects of 8-anilino-1-naphthalenesulfonate, NiCl2, and uranyl acetate on the responses to anodal current were similar to those on the responses to chemical stimulation. (c) Anodal stimulation of the tongue adapted to 50 mM CaCl2 resulted in a large response, whereas application of 1 M CaCl2 to the tongue adapted to 50 mM CaCl2 produced only a small response. This, together with theoretical considerations, suggested that the accumulation of salts on the tongue surface is not the cause of the generation of the response to anodal current. (d) Cathodal current suppressed the responses induced by 1 mM CaCl2, 0.3 M ethanol, and distilled water. (e) The addition of EGTA or Ca-channel blockers (CdCl2 and verapamil) to the perfusing solution of the lingual artery reversibly suppressed both the responses to chemical stimulus (NaCl) and to anodal current with 10 mM NaCl. (f) We assume from the results obtained that electrical current from the microvillus membrane of a taste cell to the synaptic area supplied by anodal stimulation or induced by chemical stimulation activates the voltage-dependent Ca channel at the synaptic area.     

Potassium modulation of taurine transport across the frog retinal pigment epithelium

Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.     

How the anisotropy of the intracellular and extracellular conductivities influences stimulation of cardiac muscle

The bidomain model, which describes the behavior of many electrically active tissues, is equivalent to a multi-dimensional cable model and can be represented by a network of resistors and capacitors. For a two-dimensional sheet of tissue, the intracellular and extracellular conductivity tensors can be visualized as two ellipses. For any pair of conductivity tensors, a coordinate transformation can be found that reduces the extracellular ellipse to a circle and aligns the intracellular ellipse with the coordinate axes. The eccentricity of the intracellular ellipse in this new coordinate system is an important parameter. It can have two special values: zero (in which case the tissue has equal anisotropy ratios) or one (in which case the tissue is comprised of one-dimensional fibers coupled through the two-dimensional extracellular space). Thus the bidomain model provides a unifying framework within which the electrical behavior of a wide variety of nerve and muscle tissues can be studied.When the anisotropy ratios in the intracellular and extracellular domains are not equal, stimulation with an anode always causes depolarization of some region of tissue. An analogous effect occurs in models that describe one-dimensional fibers, in which an activating function determines the site of stimulation. Experiments indicate that cardiac muscle does not have equal anisotropy ratios. Therefore, models developed to describe stimulation of axons may also help in understanding stimulation of two- or three-dimensional cardiac tissue, and may explain the concept of anodal stimulation of cardiac tissue through a virtual cathode.     

Cardiac excitation mechanisms, wavefront dynamics and strength-interval curves predicted by 3D orthotropic bidomain simulations

The assessment and understanding of cardiac excitation mechanisms is very important for the development and improvement of implantable cardiac devices, pacing protocols, and arrhythmia treatments. Previous bidomain simulation studies have investigated cathodal and anodal make/break mechanisms of cardiac excitation and strength-interval (S-I) curves in two-dimensional sheets or cylindrical domains, that by symmetry reduce to the two-dimensional case. In this work, cathodal and anodal S-I curves are studied by means of detailed bidomain simulations which include: (i) three-dimensional cardiac slabs; (ii) transmural fiber rotation; (iii) unequal orthotropic anisotropy of the conducting media; (iv) incorporation of funny and electroporation currents in the ventricular membrane model. The predicted shape of cathodal and anodal S-I curves exhibit the same features of the S-I curves observed experimentally and the break/make transition coincides with the final descending phase of the S-I curves. Away from the break/make transition, only the break or make excitation mechanism is observed independently of the stimulus strength, whereas within an interval at the break/make transition, new paradoxical excitation behaviors are observed that depend on the stimulus strength.     

Extracellular calcium transients and action potential configuration changes related to post-stimulatory potentiation in rabbit atrium

Extracellular calcium transients were monitored with 2 mM tetramethylmurexide at low calcium (250 microM total, 130 microM free), and action potentials were monitored together with developed tension at normal calcium (1.3 mM) during the production and decay of post-stimulatory potentiation in rabbit left atrial strips. At normal calcium, the contractile potentiation produced by a brief burst of 4 Hz stimulation is lost in three to five post-stimulatory excitations, which correlate with a negative staircase of the late action potential. At low calcium, stimulation at 4 Hz for 3-8 s results in a net extracellular calcium depletion of 5-15 microM. At the subsequent potentiated contraction (1-45 s rest), total extracellular calcium increases by 4-8 microM. The contractile response at a second excitation is greatly suppressed and results in little or no further calcium shift; the sequence can be repeated immediately thereafter. Reducing external sodium to 60 mM (sucrose replacement) enhances post-rest contractions, suppresses the late action potential, nearly eliminates loss of contractility and net calcium efflux at post-rest excitations, and markedly reduces extracellular calcium depletion during rapid stimulation. 4-Aminopyridine (1 mM) markedly suppresses the rapid early repolarization of this preparation at post-rest excitations and the loss of contractility at post-rest stimulation from the rested state; during a post-stimulatory potentiation sequence at low calcium, replenishment of extracellular calcium takes several post-stimulatory excitations. Ryanodine (10 nM to 5 microM) abolishes the post-stimulatory contraction at rest periods of greater than 5 s. If the initial repolarization is rapid, ryanodine suppresses the late action potential, calcium efflux during quiescence is greatly accelerated, and subsequent excitations do not result in an accumulation of extracellular calcium. A positive staircase of the early action potential correlates with the magnitude of net extracellular calcium depletion. These findings demonstrate that negative contractile staircases at post-rest stimulation correspond closely to an accumulation of extracellular calcium at activation and a negative staircase of the late action potential; the correlation of these three events suggests that electrogenic sodium-calcium exchange is the common underlying mechanism.     

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.     

Burn trauma alters calcium transporter protein expression in the heart.

We have shown previously that burn trauma produces significant cardiac dysfunction, which is first evident 8 h postburn and is maximal 24 h postburn. Because calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism by which burn injury may cause cardiac abnormalities is via calcium dyshomeostasis. We hypothesized that major burn injury alters cardiomyocyte calcium handling through changes in calcium transporter expression. Sprague-Dawley rats were given either burn injury or no burn injury (controls). Cardiomyocyte intracellular calcium and sodium were quantified at various times postburn by fura 2-AM or sodium-binding benzofuran isophthalate fluorescent indicators, respectively. In addition, hearts freeze-clamped at various times postburn (2, 4, 8, and 24 h) were used for Western blot analysis using antibodies against the sarcoplasmic reticulum calcium-ATPase (SERCA), the L-type calcium-channel, the ryanodine receptor, the sodium/calcium exchanger, or the sodium-potassium-ATPase. Intracellular calcium levels were elevated significantly 8-24 h postburn, and intracellular sodium was increased significantly 4 through 24 h postburn. Expression of SERCA was significantly reduced 1-8 h postburn, whereas L-type calcium-channel expression was diminished 1 and 2 h postburn (P < 0.05) but returned toward control levels 4 h postburn. Ryanodine receptor protein was significantly reduced at 1 and 2 h postburn, returning to baseline by 4 h postburn. Sodium/calcium exchanger expression was significantly elevated 2 h postburn but was significantly reduced 24 h postburn. An increase in sodium-potassium-ATPase expression occurred 2-24 h postburn. These data confirm that burn trauma alters calcium transporter expression, likely contributing to cardiomyocyte calcium loading and cardiac contractile dysfunction.     

Activation of specific membrane mechanisms in the myocardium cells on early stages of ischemia

Electron probe microanalysis was employed to determine the elemental concentration (K,Na,Cl) in a myocyte on cryosections of the papillary muscle of the isolated rat (Wistar) heart. Protocols of global ischemia and ischemic conditions under glucose-free anoxic perfusion were applied. It was shown that global ischemia induces potassium deficiency (94 +/- 2 mM) in the myocyte and an increase in the level of sodium (72 +/- 4 mM) and chlorine (42 +/- 1 mM) in the cytoplasm compared with intact cell (122 +/- 2; 36 +/- 1; 24 +/- 1 mM). Glucose-free anoxic perfusion leads to a smooth fall of potassium concentration in the cell up to 54 +/- 2 mM with the retention of intracellular sodium (40 +/- 1 mM) and chlorine (26 +/- 1 mM) level. The present finding suggest that, in early ischemia, specific membrane mechanisms of ion transport are activated. Among these are KNa channel, Hi(+)-Nao+ exchange, KATP channel, lactate transport from the cell, associated either with potassium efflux to the extracellular space or chlorine influx into the myocyte. It is assumed that Na/K-ATPase is also activated under ischemic conditions.     

The Spread of Excitation in the Embryonic Chick Heart

The spread of excitation in embryonic chick hearts, ranging in age from 7 to 20 days, was studied with both intracellular and extracellular electrodes. Evidence that the delay in ventricular excitation could be attributed to the cells of the entire atrioventricular (AV) ring was obtained, in part, from sagittal sections of the heart. In the intact preparation, uniform propagation occurred throughout the atrial roof at an apparent conduction velocity of 0.4 to 0.5 meter/sec. Delay of impulse propagation was localized in a very narrow band of tissue which extended across the AV ring. The apparent conduction velocity of this tissue was between 0.003 and 0.005 meter/sec. Both normal and retrograde propagation revealed the spread of conduction across the AV ring to be decremental in nature. This finding was supported by high frequency stimulation experiments which gave rise to AV block localized in the cells of the AV ring. Cardiac rhythmicity and AV transmission were responsive to acetylcholine and norepinephrine in much the same manner as in the adult mammalian heart. The present findings are in support of the hypothesis that the embryonic AV ring is the functional counterpart of the adult AV node.