Vulnerable window for conduction block in a one-dimensional cable of cardiac cells, 2: multiple extrasystoles

Unidirectional conduction block of premature extrasystoles can lead to initiation of cardiac reentry, causing lethal arrhythmias including ventricular fibrillation. Multiple extrasystoles are often more effective at inducing unidirectional conduction block and reentry than a single extrasystole. Since the substrate for conduction block is spatial dispersion of refractoriness, in this study we investigate how the first extrasystole modulates this dispersion to influence the "vulnerable window" for conduction block by subsequent extrasystoles, particularly in relation to action potential duration restitution and conduction velocity restitution properties. Using a kinematic model to represent wavefront-waveback interactions and simulations with the Luo-Rudy model in a one-dimensional cable of cardiac cells, we show that in homogeneous tissue, a premature extrasystole can create a large dispersion of refractoriness leading to conduction block of a subsequent extrasystole. In heterogeneous tissue, however, a premature extrasystole can either reduce or enhance the dispersion of refractoriness depending on its propagation direction with respect to the previous beat. With multiple extrasystoles at random coupling intervals, vulnerability to conduction block is proportional to their number. In general, steep action potential duration restitution and broad conduction velocity restitution promote dispersion of refractoriness in response to multiple extrasystoles, and thus enhance vulnerability to conduction block. These restitution properties also promote spatially discordant alternans, a setting which is particularly prone to conduction block. The equivalent dispersion of refractoriness created dynamically in homogeneous tissue by spatially discordant alternans is more likely to cause conduction block than a comparable degree of preexisting dispersion in heterogeneous tissue.     

Mechanoelectric feedback leads to conduction slowing and block in acutely dilated atria: a modeling study of cardiac electromechanics

Atrial fibrillation, a common cardiac arrhythmia, is promoted by atrial dilatation. Acute atrial dilatation may play a role in atrial arrhythmogenesis through mechanoelectric feedback. In experimental studies, conduction slowing and block have been observed in acutely dilated atria. In the present study, the influence of the stretch-activated current (I(sac)) on impulse propagation is investigated by means of computer simulations. Homogeneous and inhomogeneous atrial tissues are modeled by cardiac fibers composed of segments that are electrically and mechanically coupled. Active force is related to free Ca(2+) concentration and sarcomere length. Simulations of homogeneous and inhomogeneous cardiac fibers have been performed to quantify the relation between conduction velocity and I(sac) under stretch. In our model, conduction slowing and block are related to the amount of stretch and are enhanced by contraction of early-activated segments. Conduction block can be unidirectional in an inhomogeneous fiber and is promoted by a shorter stimulation interval. Slowing of conduction is explained by inactivation of Na(+) channels and a lower maximum upstroke velocity due to a depolarized resting membrane potential. Conduction block at shorter stimulation intervals is explained by a longer effective refractory period under stretch. Our observations are in agreement with experimental results and explain the large differences in intra-atrial conduction, as well as the increased inducibility of atrial fibrillation in acutely dilated atria.     

Embryonic stem cells form an organized, functional cardiac conduction system in vitro

A functional pacemaking-conduction system is essential for maintaining normal cardiac function. However, no reproducible model system exists for studying the specialized cardiac pacemaking-conduction system in vitro. Although several molecular markers have been shown to delineate components of the cardiac conduction system in vivo, the functional characteristics of the cells expressing these markers remain unknown. The ability to accurately identify cells that function as cardiac pacemaking cells is crucial for being able to study their molecular phenotype. In differentiating murine embryonic stem cells, we demonstrate the development of an organized cardiac pacemaking-conduction system in vitro using the coexpression of the minK-lacZ transgene and the chicken GATA6 (cGATA6) enhancer. These markers identify clusters of pacemaking "nodes" that are functionally coupled with adjacent contracting regions. cGATA6-positive cell clusters spontaneously depolarize, emitting calcium signals to surrounding contracting regions. Physically separating cGATA6-positive cells from nearby contracting regions reduces the rate of spontaneous contraction or abolishes them altogether. cGATA6/minK copositive cells isolated from embryoid cells display characteristics of specialized pacemaking-conducting cardiac myocytes with regard to morphology, action potential waveform, and expression of a hyperpolarization-activated depolarizing current. Using the cGATA6 enhancer, we have isolated cells that exhibit electrophysiological and genetic properties of cardiac pacemaking myocytes. Using molecular markers, we have generated a novel model system that can be used to study the functional properties of an organized pacemaking-conducting contracting system in vitro. Moreover, we have used a molecular marker to isolate a renewable population of cells that exhibit characteristics of cardiac pacemaking myocytes.     

Evidence for a single pool of myo-inositol in hormone-responsive WRK-1 cells

Previous reports have suggested the existence of at least two pools of cellular myo-inositol (Ins); it has been further hypothesized that only one of these pools is utilized during hormone-activated, cyclic phosphatidylinositol (PtdIns) resynthesis. In an effort to investigate this possibility, we have undertaken kinetic studies of Ins metabolism in WRK-1 cells. Our results indicate that a single pool of Ins is involved in both basal and activated PtdIns synthesis. Ins generated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) mixes with the existing pool of free Ins and is not used exclusively for resynthesis of PtdIns. © 1995 Wiley-Liss, Inc.     

Generality of a power-law long-term correlation in beat timings of single cardiac cells

Statistical properties of spontaneous contractions of atrial muscle cells were examined and compared to those of ventricular muscle cells. The cells derived from atria of neonatal rats exhibit spindle morphology, and they were found to express α-smooth muscle actin and hyperpolarization-activated cation channel 4, both of which are known marker of neonatal atrial muscle cells. The short-term properties of spontaneous contractions of atrial cells, characterized by considerably large beat rate and absence of bursts, are distinct from those of ventricular muscle cells. Despite of these differences, the long-term properties of the beat-rate fluctuations exhibit a remarkable similarity to those of ventricular cells. In particular, the presence of power-law correlation characterized as 1/f^β noise (β ≈ 1) was also confirmed for atrial cells for the first time. The observed similarity of the long-term characteristics of beat-rate fluctuation suggests the presence of a general regulatory mechanism of the cellular function.     

Notch1b and neuregulin are required for specification of central cardiac conduction tissue

Normal heart function is critically dependent on the timing and coordination provided by a complex network of specialized cells: the cardiac conduction system. We have employed functional assays in zebrafish to explore early steps in the patterning of the conduction system that previously have been inaccessible. We demonstrate that a ring of atrioventricular conduction tissue develops at 40 hours post-fertilization in the zebrafish heart. Analysis of the mutant cloche reveals a requirement for endocardial signals in the formation of this tissue. The differentiation of these specialized cells, unlike that of adjacent endocardial cushions and valves, is not dependent on blood flow or cardiac contraction. Finally, both neuregulin and notch1b are necessary for the development of atrioventricular conduction tissue. These results are the first demonstration of the endocardial signals required for patterning central ;slow' conduction tissue, and they reveal the operation of distinct local endocardial-myocardial interactions within the developing heart tube.     

Bacterial lipopolysaccharide induces a conduction block in the sciatic nerves of rats

A single injection of Escherichia coli lipopolysaccharide (LPS; intraperitoneally [i.p.] and intravenously [i.v.]) reliably induces peripheral nerve disturbances in the hindlimbs of inbred Australian albino Wistar (AaW) rats. In the series of experiments presented here, we aimed to characterize this syndrome by examining electrophysiologic, immunologic, and immunochemical features. The LPS-induced neurologic sequelae in AaW rats were transient, at least partly reversible by drug treatment, and were not associated with any detectable neuropathologic findings by light microscopy. Neurologic sequelae were prevented by administration of dexamethasone and by pretreatment with the macrophage inhibitor gadolinium chloride, suggesting that they were caused by LPS-induced activation of peripheral macrophages. Sequelae were associated with early decreases in compound muscle-action potential amplitudes, indicating impaired functioning of either proximal sciatic nerve axons and/or neuromuscular synapses. Spinal somatosensory-evoked potential latencies also were increased, indicating impaired somatosensory function at the sciatic nerve, dorsal roots, spinal cord, and/or postsynaptic interneurons, although the precise location of impairment could not be delineated. Similarities between this syndrome and immune-mediated polyneuropathies in humans are discussed.     

Selective block of calcium current by lanthanum in single bullfrog atrial cells

A single suction microelectrode voltage-clamp technique was used to study the actions of lanthanum ions (La3+) on ionic currents in single cells isolated from bullfrog right atrium. La3+, added as LaCl3, blocked the "slow" inward Ca2+ current (ICa) in a dose-dependent fashion; 10(-5) M produced complete inhibition. This effect was best fitted by a dose-response curve that was calculated assuming 1:1 binding of La3+ to a site having a dissociation constant of 7.5 x 10(-7) M. La3+ block was reversed (to 90% of control ICa) following washout and, in the presence of 10(-5) M La3+, was antagonized by raising the Ca2+ concentration from 2.5 to 7.5 mM (ICa recovered to 56% of the control). However, the latter effect took approximately 1 h to develop. Concentrations of La3+ that reduced ICa by 12-67%, 0.1-1.5 x 10(-6) M, had no measurable effect upon the voltage dependence of steady state ICa inactivation, which suggest that at these concentrations there are no significant surface-charge effects of La3+ on this gating mechanism. Three additional findings indicate that doses of La3+ that blocked ICa failed to produce nonspecific effects: (a) 10(-5) M La3+ had no measurable effect on the time-independent inwardly rectifying current, IK1; (b) the same concentration had no effect on the kinetics, amplitude, or voltage dependence of a time- and voltage-dependent K+ current, IK; and (c) 10(-4) M La3+ did not alter the size of the tetrodotoxin-sensitive inward Na+ current, INa, or the voltage dependence of its steady state inactivation. Higher concentrations (0.5-1.0 mM) reduced both IK1 and IK, and shifted the steady state activation curve for IK toward more positive potentials, presumably by reducing the external surface potential. Our results suggest that at a concentration of less than or equal to 10(-5) M, La3+ inhibits ICa selectively by direct blockade of Ca channels rather than by altering the external surface potential. At higher concentrations, La3+ exhibits nonspecific effects, including neutralization of negative external surface charge and inhibition of other time- and voltage-dependent ionic currents.     

Nonsteady motion in unloaded contractions of single frog cardiac cells.

We studied the mode of shortening of enzymatically isolated single frog cardiac cells with a high-speed videosystem to see whether or not shortening is smooth. The segmental shortening of the cell in response to electrical stimulation exhibited a clear pause following the initial shortening over a distance of approximately 11 nm/half-sarcomere. Several preparations showed a second pause following the initial one. Nonsteady motion with a pause lasted usually a few tens of milliseconds. The duration of nonsteady motion was shorter in cells with large velocities of steady shortening following the pause than those with smaller velocities.     

Translocation mechanism of Na-Ca exchange in single cardiac cells of guinea pig

We have studied in single cardiac ventricular cells of guinea pig the ionic translocation mechanism of the electrogenic Na-Ca exchange, i.e., whether Na and Ca ions countercross the membrane simultaneously or consecutively with "ping pong" kinetics. The dose-response relation between the external Ca concentrations [( Ca]o) and the current density of the outward Na-Ca exchange current were measured at three different intracellular Na concentrations [( Na]i) in the absence of external Na. Nonlinear regression curves of the dose-response relation obtained by computer revealed Michaelis-Menten type hyperbola from which the [Ca]o giving a half-maximal response (apparent KmCao or K'mCao) and the apparent maximum current magnitude (I'max) were estimated at each [Na]i. As [Na]i increased, the K'mCao increased progressively and the value of K'mCao/I'max tended to decrease. These results are consistent with the simultaneous mechanism. The K'mCao/I'max values, however, were small and close to each other, so it was not possible to completely preclude a consecutive mechanism.     

A simple model of delay,block and one way conduction in Purkinje fibers

Summary A simple qualitative model of the cardiac Purkinje fiber is introduced for the purpose of numerical simulation of experiments on conduction of the cardiac impulse. Well known approximation techniques are used to illustrate the behavior of the model membrane in the cases of space clamp and propagation of traveling pulses in a uniform infinite fiber. The results of the numerical simulations are then presented, and shown to be comparable to experiment.     

A single red-light pulse leads to a block of greening in the high-pigment-1 mutant of tomato

A single pulse of red light (R) given to 4-d-old etiolated high-pigment-1 (hp-1) mutant tomato (Solanum lycopersicum L.) seedlings followed by a 3-d dark period is demonstrated to result in a block of greening in subsequent white light. Wild-type seedlings green normally under this regime. The block of greening in the hp-1 mutant depends on the length of the dark period before and after the R pulse and operates via the low-fluence-response mode of phytochrome action. This block of greening takes place in hp-1 double mutants lacking either phytochrome A or phytochrome B1, but is absent in the hp-1 triple mutant lacking both phytochromes A and B1. These observations enable a screen to be devised for new phytochrome B1 mutants either within the photoreceptor or mutants defective in phytochrome B1-signalling steps which result in loss of capacity to green, by mutagenising the phytochrome A-deficient hp-1, fri double mutant. Received: 20 February 1998 / Accepted: 18 June 1998     

A novel mutant allele of Ncx1: a single amino acid substitution leads to cardiac dysfunction

The biological role and structure-function relationship of the Na(+)Ca(2+) exchanger NCX1 have been the subject of much investigation. Subtle mutagenesis to study the function of a protein seems only feasible in in vitro systems, but genetic forward screens have the potential to provide in vivo models to study single amino acid substitutions. In a genetic screen in mouse, we have isolated a mutant line carrying a novel mutant allele of the mouse Ncx1 gene. In this allele, a point mutation causes the substitution of a highly conserved asparagine residue (N874) with lysine. Accepted models for NCX1 structure propose that the affected amino acid is located in one of the reentrant membrane loops and experiments in vitro have identified N874 as critical for the ion transport function of NCX1. We found severe circulation defects and defective placentation in homozygous Ncx1(N87K4) mutant embryos, making the phenotype essentially indistinguishable from those of previously described null mutants. By ex vivo analysis, we demonstrated intrinsic functional abnormalities of cardiomyocytes. Western blot analysis and immunohistochemistry demonstrated normal levels and subcellular localization of the altered protein, ruling out the possibility that the abnormalities are a mere consequence of a major disturbance of protein structure. This study confirms and extends studies in vitro indicating the significance of amino acid N874 for the function of the NCX1 protein. It provides an in vivo model for this mutation and demonstrates the potential of forward genetic screens in a mammalian system.     

Fenestration: a window of opportunity for carnivorous plants

A long-standing but controversial hypothesis assumes that carnivorous plants employ aggressive mimicry to increase their prey capture success. A possible mechanism is that pitcher plants use aggressive mimicry to deceive prey about the location of the pitcher''s exit. Specifically, species from unrelated families sport fenestration, i.e. transparent windows on the upper surfaces of pitchers which might function to mimic the exit of the pitcher. This hypothesis has not been evaluated against alternative hypotheses predicting that fenestration functions to attract insects from afar. By manipulating fenestration, we show that it does not increase the number of Drosophila flies or of two ant species entering pitchers in Sarracenia minor nor their retention time or a pitcher''s capture success. However, fenestration increased the number of Drosophila flies alighting on the pitcher compared with pitchers of the same plant without fenestration. We thus suggest that fenestration in S. minor is not an example of aggressive mimicry but rather functions in long-range attraction of prey. We highlight the need to evaluate aggressive mimicry relative to alternative concepts of plant–animal communication.     

Induction of a reversible G 1 block in human glia-like cells by cytochalasin B

In cultures of normal adult human glia-like cells, density-dependent cell cycle inhibition (topoinhibition) and contact inhibition of ruffling occur almost simultaneously, suggesting a functional coupling between activities of the cell surface and the initiation of DNA synthesis. The present paper examines whether cytochalasin B (CB), which reversibly inhibits ruffling, also blocks the glia cell cycle.The effects of the drug (2 μg/ml) were the following:

1. 1. Initiation of DNA synthesis of subcultivated stationary cells was inhibited.

2. 2. Stimulation of DNA synthesis in stationary cells by medium change was suppressed.

3. 3. Migration of cells into a wound in a confluent cell layer was blocked as well as the initiation of DNA synthesis in cells lining the wound.

4. 4. Initiation (but not continuation) of DNA synthesis of exponentially growing cells was inhibited leading to a population mainly arrested in G 1 as determined by microspectrophotometry on Feulgen-stained cells. Topoinhibited cells were also blocked in G 1. Since cytokinesis was blocked by CB, a fraction of binuclear cells appeared.

The cell cycle block induced by CB was reversible, even after several weeks of treatment, with the exception that binuclear cells more reluctantly entered the S phase after release of the block.In conclusion, CB efficiently induces a reversible and probably physiologic cell cycle block. This finding strengthens the notion of a connection between cell membrane and cell proliferation. The underlying mechanism is discussed.     

Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells

The Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6 h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells.     

Turnover of nuclear RNA in HeLa cells: evidence for a single ribonucleotide pool

The role of the ribonucleotide pool in measuring turn-over of RNA has been studied. Evidence presented previously has assumed a single pool. The nucleotide base ratios of two different species of nuclear RNA (heterogeneously sedimenting nuclear RNA and ribosomal precursor RNA) are known. We have studied the rate of approach to the equilibrium base ratio established by labeling, either via the salvage pathway or the endogenous route. In either case, normalization of the measured U:C ratios is accomplished by dividing the equilibrium U:C ratio in an isolated species by the measured base ratio (specific U:C ratio). These studies are consistent with the existence of a single ribonucleotide pool.     

Fractional cable equation models for anomalous electrodiffusion in nerve cells: infinite domain solutions

We introduce fractional Nernst-Planck equations and derive fractional cable equations as macroscopic models for electrodiffusion of ions in nerve cells when molecular diffusion is anomalous subdiffusion due to binding, crowding or trapping. The anomalous subdiffusion is modelled by replacing diffusion constants with time dependent operators parameterized by fractional order exponents. Solutions are obtained as functions of the scaling parameters for infinite cables and semi-infinite cables with instantaneous current injections. Voltage attenuation along dendrites in response to alpha function synaptic inputs is computed. Action potential firing rates are also derived based on simple integrate and fire versions of the models. Our results show that electrotonic properties and firing rates of nerve cells are altered by anomalous subdiffusion in these models. We have suggested electrophysiological experiments to calibrate and validate the models.