Effect of Transmural Differences in Excitation-Contraction Delay and Contraction Velocity on Left Ventricle Isovolumic Contraction: A Simulation Study.

Recent studies have shown that left ventricle (LV) exhibits considerable transmural differences in active mechanical properties induced by transmural differences in electrical activity, excitation-contraction coupling, and contractile properties of individual myocytes. It was shown that the time between electrical and mechanical activation of myocytes (electromechanical delay: EMD) decreases from subendocardium to subepicardium and, on the contrary, the myocyte shortening velocity (MSV) increases in the same direction. To investigate the physiological importance of this inhomogeneity, we developed a new finite element model of LV incorporating the observed transmural gradients in EMD and MSV. Comparative simulations with the model showed that when EMD or MSV or both were set constant across the LV wall, the LV contractility during isovolumic contraction (IVC) decreased significantly ((dp/dt)max⁡  was reduced by 2 to 38% and IVC was prolonged by 18 to 73%). This was accompanied by an increase of transmural differences in wall stress. These results suggest that the transmural differences in EMD and MSV play an important role in physiological contractility of LV by synchronising the contraction of individual layers of ventricular wall during the systole. Reduction or enhancement of these differences may therefore impair the function of LV and contribute to heart failure.

Different Densities of Na-Ca Exchange Current in T-Tubular and Surface Membranes and Their Impact on Cellular Activity in a Model of Rat Ventricular Cardiomyocyte.

The ratio of densities of Na-Ca exchanger current (INaCa) in the t-tubular and surface membranes (INaCa-ratio) computed from the values of INaCa and membrane capacitances (Cm) measured in adult rat ventricular cardiomyocytes before and after detubulation ranges between 1.7 and 25 (potentially even 40). Variations of action potential waveform and of calcium turnover within this span of the INaCa-ratio were simulated employing previously developed model of rat ventricular cell incorporating separate description of ion transport systems in the t-tubular and surface membranes. The increase of INaCa-ratio from 1.7 to 25 caused a prolongation of APD (duration of action potential at 90% repolarisation) by 12, 9, and 6% and an increase of peak intracellular Ca2+ transient by 45, 19, and 6% at 0.1, 1, and 5 Hz, respectively. The prolonged APD resulted from the increase of INaCa due to the exposure of a larger fraction of Na-Ca exchangers to higher Ca2+ transients under the t-tubular membrane. The accompanying rise of Ca2+ transient was a consequence of a higher Ca2+ load in sarcoplasmic reticulum induced by the increased Ca2+ cycling between the surface and t-tubular membranes. However, the reason for large differences in the INaCa-ratio assessed from measurements in adult rat cardiomyocytes remains to be explained.

Effect of ethanol and acetaldehyde at clinically relevant concentrations on atrial inward rectifier potassium current IK1: separate and combined effect.

Atrial fibrillation is the most common arrhythmia at alcohol consumption. Its pathogenesis is complex, at least partly related to changes of cardiac inward rectifier potassium currents including IK1. Both ethanol and acetaldehyde have been demonstrated to considerably modify IK1 in rat ventricular myocytes. However, analogical data on the atrial IK1 are lacking. The present study aimed to analyse IK1 changes induced by ethanol and acetyldehyde in atrial myocytes. The experiments were performed by the whole cell patch-clamp technique at 23 ± 1°C on enzymatically isolated rat and guinea-pig atrial myocytes as well as on expressed human Kir2.3 channels. Ethanol (8 - 80 mM) caused a dual effect on the atrial IK1 showing the steady-state activation in some cells but inhibition in others in agreement with the ventricular data; on average, the activation was observed (at 20 mM by 4.3 and 4.5% in rat and guinea-pig atrial myocytes, respectively). The effect slightly increased with depolarization above -60 mV. In contrast, the current through human Kir2.3 channels (prevailing atrial IK1 subunit) was inhibited in all measured cells. Unlike ethanol, acetaldehyde (3 µM) markedly inhibited the rat atrial IK1 (by 15.1%) in a voltage-independent manner, comparably to the rat ventricular IK1. The concurrent application of ethanol (20 mM) and acetaldehyde (3 µM) resulted in the steady-state IK1 activation by 2.1% on average. We conclude that ethanol and even more acetaldehyde affected IK1 at clinically relevant concentrations if applied separately. Their combined effect did not significantly differ from the effect of ethanol alone.

Analgesia epidural continua para trabajo de parto en una paciente con polineuropatía desmielinizante periférica / Continuous epidural analgesia for labour and delivery in a patient with demyelinating peripheral polyneuropathy

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Prevalence of generalised joint hypermobility in school-aged children from east-central European region.

BACKGROUND: There is no literature regarding joint mobility in children of the Central and Eastern Europe. Studies describing clinical characteristics and functional outcomes are still needed. The aim of this study was to assess the prevalence of generalised joint hypermobility (GJH) in the group of school-aged children from Vilnius, the capital city of Lithuania, in relation to different cut-off values of the Beighton score (BS), and to identify possible patients with joint hypermobility syndrome. MATERIALS AND METHODS: The representative sample of this study was calculated to be 760 subjects. A total of 778 children from different schools were screened for the mobility of joints. The medical examination included an assessment of joints' hypermobility according to the BS. The presence of specific signs (marfanoid habitus, antimongoloid slant and drooping eyelids) was assessed additionally. Parents of all involved children were asked to answer the questions developed based on the Brighton criteria regarding the medical history of children. RESULTS: The prevalence of GJH in school-aged children from Vilnius, depending on the BS cut-off value, was 19.2% (BS ≥ 4), 9.5% (BS ≥ 5) or 5.7% (BS ≥ 6). The increased range of mobility was most frequently detected in thumbs of school- -aged children. The frequency of hyperextension > 10o in knees was 7- to 8-fold lower than the frequency of hyperextension > 10o in a passive opposition of the thumb. The evaluation results were similar on the left and right sides in 87.4% cases of thumb opposition, 90.1% cases of hyperextension of 5th finger, 87.9% cases of elbow manoeuvres, and 94.8% attempts to hyperextend knee. CONCLUSIONS: The prevalence of GJH in school-aged children from Vilnius depends on the BS cut-off value and ranges from 5.7% to 19.2%.

Dual effect of ethanol on inward rectifier potassium current IK1 in rat ventricular myocytes.

Alcohol consumption may result in electrocardiographic changes and arrhythmias. Important role of modifications of the inward rectifier potassium current I(K1) in arrhythmogenesis is well established. Considering lack of relevant data, we aimed at studying the effect of 0.2-200 mM ethanol on I(K1) in enzymatically isolated rat right ventricular myocytes using the whole cell patch-clamp technique at 23±1°C. Ethanol reversibly affected I(K1) in a dual way. At a very low concentration of 0.8 mM (≈~0.004%), ethanol significantly decreased IK1 by 6.9±2.7%. However, at concentrations of ethanol ≥20 mM (≈0.09%), I(K1) was conversely significantly increased (by 16.6±4.0% at 20 mM and 24.5±2.4% at 80 mM). The steady-state I(K1) increase was regularly preceded by its transient decrease at the beginning of ethanol application. Under 2 and 8 mM ethanol, I(K1) was decreased at the steady-state in some cells but increased in others. Both effects were voltage-independent. In agreement with the observed effects of ethanol on I(K1), a transient action potential (AP) prolongation followed by its final shortening were observed after the application of ethanol in a low concentration of 8 mM (≈0.04%). Under the effect of 0.8 mM ethanol, only AP prolongation was apparent which agreed well with the above described I(K1) decrease. Other AP characteristics remained unaltered in both concentrations. These observations corresponded with the results of mathematical simulations in a model of the rat ventricular myocyte. To summarize, changes of the cardiac I(K1) under ethanol at concentrations relevant to the current alcohol consumption were first demonstrated in ventricular myocytes in this study. The observed dual ethanol effect suggests at least two underlying mechanisms that remain to be clarified. The ethanol-induced I(K1) changes might contribute to the reported alterations of cardiac electrophysiology related to alcohol consumption.

Role of t-tubules in the control of trans-sarcolemmal ion flux and intracellular Ca2+ in a model of the rat cardiac ventricular myocyte.

The t-tubules of mammalian ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell, with restricted diffusion to the bulk extracellular space. The trans-sarcolemmal flux of many ions, including Ca(2+), occurs predominantly across the t-tubule membrane and thus into and out of this restricted diffusion space. It seems possible, therefore, that ion concentration changes may occur in the t-tubule lumen, which would alter ion flux across the t-tubule membrane. We have used a computer model of the ventricular myocyte, incorporating a t-tubule compartment and experimentally determined values for diffusion between the t-tubule lumen and bulk extracellular space, and ion fluxes across the t-tubule membrane, to investigate this possibility. The results show that influx and efflux of different ion species across the t-tubule membrane are similar, but not equal. Changes of ion concentration can therefore occur close to the t-tubular membrane, thereby altering trans-sarcolemmal ion flux and thus cell function, although such changes are reduced by diffusion to the bulk extracellular space. Slowing diffusion results in larger changes in luminal ion concentrations. These results provide a deeper understanding of the role of the t-tubules in normal cell function, and are a basis for understanding the changes that occur in heart failure as a result of changes in t-tubule structure and ion fluxes.

Effect of ethanol on action potential and ionic membrane currents in rat ventricular myocytes.

AIM: Even though alcohol intoxication is often linked to arrhythmias, data describing ethanol effect on cardiac ionic channels are rare. In addition, ethanol is used as a solvent of hydrophobic compounds in experimental studies. We investigated changes of the action potential (AP) configuration and main ionic membrane currents in rat cardiomyocytes under 20-1500 m(M) ethanol. METHODS: Experiments were performed on enzymatically isolated rat right ventricular myocytes using the whole cell patch-clamp technique at room temperature. RESULTS: Ethanol reversibly decelerated the upstroke velocity and decreased AP amplitude and duration at 0.2 and 3 Hz. The fast sodium current I(Na) , l-type calcium current I(Ca) and transient outward potassium current I(to) were reversibly inhibited in a concentration-dependent manner (50% inhibition at 446 ± 12, 553 ± 49 and 1954 ± 234 m(M), respectively, with corresponding Hill coefficients 3.1 ± 0.3, 1.1 ± 0.2 and 0.9 ± 0.1). Suppression of I(Na) and I(Ca) magnitude was slightly voltage dependent. The effect on I(Ca) and I(to) was manifested mainly as an acceleration of their apparent inactivations with a decreased slow and fast time constant respectively. As a consequence of marked differences in n(H) , sensitivity of the currents to ethanol at 10% inhibition decreases in the following order: I(Ca) (75 mm, 3.5‰), I(to) (170 m(M), 7.8‰) and I(Na) (220 m(M), 10.1‰). CONCLUSION: Our results suggest a slight inhibition of all the currents at ethanol concentrations relevant to deep alcohol intoxication. The concentration dependence measured over a wide range may serve as a guideline when using ethanol as a solvent.

Modelling the cardiac transverse-axial tubular system.

The transverse-axial tubular system (TATS) of cardiac ventricular myocytes is a complex network of tubules that arises as invaginations of the surface membrane; it appears to form a specialised region of cell membrane that is particularly important for excitation-contraction coupling. However, much remains unknown about the structure and role of the TATS. In this brief review we use experimental data and computer modelling to address the following key questions: (i) What fraction of the cell membrane is within the TATS? (ii) Is the composition of the TATS membrane the same as the surface membrane? (iii) How good is electrical coupling between the surface and TATS membranes? (iv) What fraction of each current is within the TATS? (v) How important is the complex structure of the TATS network? (vi) What is the effect of current inhomogeneity on lumenal ion concentrations? (vii) Does the TATS contribute to the functional changes observed in heart failure? Although there are many areas in which experimental evidence is lacking, computer models provide a method to assess and predict the possible function of the TATS; such models suggest that although the surface and TATS membranes are electrically well coupled, concentration of ion flux pathways within the TATS, coupled to restricted diffusion, may result in the ionic composition in the TATS lumen being different from that in the bulk extracellular space, and varying with activity and in pathological conditions.

Quantification of t-tubule area and protein distribution in rat cardiac ventricular myocytes.

The transverse (t-) tubules of cardiac ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell. Many of the key proteins involved in excitation-contraction coupling appear to be located predominantly at the t-tubule membrane. Despite their importance, the fraction of cell membrane within the t-tubules remains unclear: measurement of cell capacitance following detubulation suggests approximately 32%, whereas optical measurements suggest up to approximately 65%. We have, therefore, investigated the factors that may account for this discrepancy. Calculation of the combinations of t-tubule radius, length and density that produce t-tubular membrane fractions of 32% or 56% suggest that the true fraction is at the upper end of this range. Assessment of detubulation using confocal and electron microscopy suggests that incomplete detubulation can account for some, but not all of the difference. High cholesterol, and a consequent decrease in specific capacitance, in the t-tubule membrane, may also cause the t-tubule fraction calculated from the loss of capacitance following detubulation to be underestimated. Correcting for both of these factors results in an estimate that is still lower than that obtained from optical measurements suggesting either that optical methods overestimate the fraction of membrane in the t-tubules, or that other, unknown, factors, reduce the apparent fraction obtained by detubulation. A biophysically realistic computer model of a rat ventricular myocyte, incorporating a t-tubule network, is used to assess the effect of the altered estimates of t-tubular membrane fraction on the calculated distribution of ion flux pathways.

Changes in action potentials and intracellular ionic homeostasis in a ventricular cell model related to a persistent sodium current in SCN5A mutations underlying LQT3.

In LQT3 patients, SCN5A mutations induce ultraslow inactivation of a small fraction of the hNav1.5 current, i.e. persistent Na+ current (IpNa). We explored the time course of effects of such a change on the intracellular ionic homeostasis in a model of guinea-pig cardiac ventricular cell [Pasek, M., Simurda, J., Orchard, C.H., Christé, G., 2007b. A model of the guinea-pig ventricular cardiomyocyte incorporating a transverse-axial tubular system. Prog. Biophys. Mol. Biol., this issue]. Sudden addition of IpNa prevented action potential (AP) repolarization when its conductance (gpNa) exceeded 0.12% of the maximal conductance of fast INa (gNa). With gpNa at 0.1% gNa, the AP duration at 90% repolarization (APD90) was initially lengthened to 2.6-fold that in control. Under regular stimulation at 1 Hz it shortened progressively to 1.37-fold control APD90, and intracellular [Na+]i increased by 6% with a time constant of 106 s. Further increasing gpNa to 0.2% gNa caused an immediate increase in APD90 to 5.7-fold that in control, which decreased to 2.2-fold that in control in 30s stimulation at 1 Hz. At this time diastolic [Na+]i and [Ca2+]i were, respectively, 34% and 52% higher than in control and spontaneous erratic SR Ca release occurred. In the presence of IpNa causing 46% lengthening of APD90, the model cell displayed arrhythmogenic behaviour when external [K+] was lowered to 5 mM from an initial value at 5.4 mM. By contrast, when K+ currents IKr and IKs were lowered in the model cell to produce the same lengthening of APD90, no proarrhythmic behaviour was observed, even when external [K+] was lowered to 2.5 mM.

Effect of ajmaline on action potential and ionic currents in rat ventricular myocytes.

The effect of ajmaline on action potential (AP) and ionic current components has been investigated in right ventricular myocytes of rat at room temperature using the whole cell patch clamp technique. Ajmaline decreased the upstroke velocity ((dV/dt)max) of AP and the AP amplitude, increased the AP duration measured at 50 and 90% repolarization, and reversibly inhibited most components of membrane ionic current in a concentration-dependent manner. The following values of IC50 and of the Hill coefficient (nH) resulted from approximation of the measured data by the Hill formula: for fast sodium current (INa) IC50=27.8+/-1.14 micromol/l and nH=1.27+/-0.25 at holding potential -75 mV, IC50=47.2+/-1.16 micromol/l and nH=1.16+/-0.21 at holding potential -120 mV; for L-type calcium current (ICa-L) IC50=70.8+/-0.09 micromol/l and n(H)=0.99+/-0.09; for transient outward potassium current (Ito) IC50=25.9+/-2.91 micromol/l and nH=1.07+/-0.15; for ATP-sensitive potassium current (IK(ATP)) IC50=13.3+/-1.1 micromol/l and nH=1.16+/-0.15. The current measured at the end of 300 ms depolarizing impulse was composed of an ajmaline-insensitive component and a component inhibited with IC50=61.0+/-1.1 micromol/l and nH=0.91+/-0.08. At hyperpolarizing voltages, ajmaline at high concentration of 300 micromol/l reduced the inward moiety of time-independent potassium current (IK1) by 36%. The results indicate that the inhibition of INa causes both the decreased rate of rise of depolarizing phase and the lowered amplitude of AP. The inhibition of Ito is responsible for the ajmaline-induced AP prolongation.

Effect of ajmaline on transient outward current in rat ventricular myocytes.

The mechanism of ajmaline-induced inhibition of the transient outward current (I(to)) has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of I(to) in a concentration-dependent, but frequency- and use-independent manner. In contrast to the single exponential time course of I(to)-inactivation in control conditions (tau(i) = 37.1 +/- 2.7 ms), the apparent inactivation was fitted by a sum of two exponentials under the effect of ajmaline with concentration-dependent fast and slow components (tau(f) = 11.7 +/- 0.8 ms, tau(s) = 57.6 +/- 2.7 ms at 10 micromol/l) suggesting block development primarily in the open channel state. An improved expression enabling to calculate the association and dissociation rate constants from the concentration dependence of tau(f) and tau(s) was derived and resulted in k(on) = 4.57 x 10(6) +/- 0.32 x 10(6) mol(-1).l.s(-1) and k(off) = 20.12 +/- 5.99 s(-1). The value of K(d) = 4.4 micromol/l calculated as k(off) / k(on) was considerably lower than IC(50) = 25.9 +/- 2.9 micromol/l evaluated from the concentration dependence of the integrals of I(to). Simulations on a simple model combining Hodgkin-Huxley type gating kinetics and drug-channel interaction entirely in open channel state agreed well with the experimental data including the difference between the K(d) and IC(50). According to the model, the fraction of blocked channels increases upon depolarization and declines if depolarization is prolonged. The repolarizing step induces recovery from block with time constant of 52 ms. We conclude that in the rat right ventricular myocytes, ajmaline is an open channel blocker with fast recovery from the block at resting voltage.

Quantitative modelling of interaction of propafenone with sodium channels in cardiac cells.

A mathematical model of the interaction of propafenone with cardiac sodium channels is based on experimental data that demonstrate use-dependent effects of the drug. The Clancy-Rudy model is applied to describe Na-channels in absence of the drug. The values of rate constants of the drug-receptor reaction are fitted to experimental data by iterative computer simulations using a genetic algorithm. The model suggests the following interpretation of available experimental results: First, drug molecules have access to the binding sites predominantly in the inactivated states. Secondly, the biphasic development of the block during depolarisation is consistent with a rapid increase due to drug binding in the fast inactivated state (rate constants k(on) = 645 micromol(-1) l s(-1), k(off) = 16.21 s(-1)) and a slow increase due to binding in the intermediate inactivated state (rate constants approximately 100-fold lower), followed by transition to the drug-occupied slow inactivated state (rate constants 0.784 and 0.921 s(-1)). Thirdly, the observed biphasic time course of recovery of I(Na) from block following restoration of the resting voltage results from simultaneous relief of block from the channels residing in the intermediate and slow inactivated states. Fourthly, the accumulation of blocked channels in the slow inactivated state is responsible for the observed use-dependent effects. Fifthly, when incorporated into a quantitative description of the electrical activity of a ventricular cell, the model predicts that propafenone (0.2 micromol l(-1)) effectively suppresses premature excitations, leaving the regular action potentials nearly unaffected.

Muscle FBPase in a complex with muscle aldolase is insensitive to AMP inhibition.

Real-time interaction analysis, using the BIAcore biosensor, of rabbit muscle FBPase-aldolase complex revealed apparent binding constant [K(Aapp)] values of about 4.4x10(8) M(-1). The stability of the complex was down-regulated by the glycolytic intermediates dihydroxyacetone phosphate and fructose 6-phosphate, and by the regulator of glycolysis and glyconeogenesis--fructose 2,6-bisphosphate. FBPase in a complex with aldolase was entirely insensitive to inhibition by physiological concentrations of AMP (I(0.5) was 1.35 mM) and the cooperativity of the inhibition was not observed. The existence of an FBPase-aldolase complex that is insensitive to AMP inhibition explains the possibility of glycogen synthesis from carbohydrate precursors in vertebrates' myocytes.

A quantitative model of the cardiac ventricular cell incorporating the transverse-axial tubular system.

The role of the transverse-axial tubular system (TATS) in electrical activity of cardiac cells has not been investigated quantitatively. In this study a mathematical model including the TATS and differential distribution of ionic transfer mechanisms in peripheral and tubular membranes was described. A model of ventricular cardiac cell described by Jafri et al. (1998) was adopted and slightly modified to describe ionic currents and Ca2+ handling. Changes of concentrations in the lumen of the TATS were computed from the total of transmembrane ionic fluxes and ionic exchanges with the pericellular medium. Long-term stability of the model was attained at rest and under regular stimulation. Depletion of Ca2+ by 12.8% and accumulation of K+ by 4.7% occurred in the TATS-lumen at physiological conditions and at a stimulation frequency of 1 Hz. The changes were transient and subsided on repolarization within 800 ms (Ca2+) and 300 ms (K+). Nevertheless, the course of action potentials remained virtually unaltered. Simulations of voltage clamp experiments demonstrated that variations in tubular ionic concentrations were detectable as modulation of the recorded membrane currents.

Quantitative analysis of cardiac electrical restitution.

Electrical restitution (ER) of cardiac cells is an aggregate of events that rhythmically restore the initial conditions of electric signal (action potential) generation. Its analysis represents an important insight into cardiac arrhythmogenesis. The aim of this work is to theoretically substantiate and verify a novel approach allowing for the quantification of the individual ionic current components of ER. A method of analysis of the primary, initial conditions-setting restitution processes (apart from the secondary, test pulse-affected ones) is proposed. Both processes are described as sums of their measurable constituents. It is demonstrated that the optimum parameter of ER is the electric charge that is transferred through ionic channels and carriers during the test impulse. The theory was tested by using voltage-clamped canine ventricular preparations and by computer simulations. The experimental ER curve of canine ventricular muscle was constructed using action potential (AP) plateau voltage and half-repolarization time as parameters. At 30 degrees C and 0.5 Hz stimulation, the ER curve peaked, on average, after 400 ms with a 10% overshoot. Of this plateau elevation, 50% was due to 4-aminopyridine-sensitive transient outward current and 44% was due to verapamil-sensitive current. The delayed outward current antagonized the overshoot by about 6%. It was found that the initial conditions (i.e. the primary restitution processes) tend to strongly alter the plateau voltage of the premature AP. However, the final deviation is by about one order less. It is concluded that the voltage-dependent secondary processes counteract the effect of the primary processes, thereby suggesting strong negative feedback control of natural APs.

Endothelial leukocyte adhesion molecule 1: direct expression cloning and functional interactions.

A cDNA for endothelial leukocyte adhesion molecule 1 (ELAM-1) was isolated by transient expression in COS-7 cells of a subtracted cDNA library from cytokine-treated human umbilical vein endothelial cells (HUVECs), with selection of ELAM-1-expressing clones by adhesion of transfected cells to the human promyelocytic cell line HL-60. This cloning method requires neither antibody nor purified ligand. ELAM-1-expressing COS cells bind the promyelocytic cell line HL-60 by a Ca2(+)-dependent but temperature-independent mechanism. Although ELAM-1 is homologous to mammalian lectins, its interaction with HL-60 cells is not inhibited by simple carbohydrate structures. ELAM-1-expressing COS cells also bind human neutrophils and the human colon carcinoma cell line HT-29, but not the B-cell line Ramos. However, Ramos cells adhere to cytokine-treated HUVECs but not control HUVECs, confirming the existence of other inducible adhesion molecules. In addition, the binding of HL-60 cells or neutrophils to ELAM-1-expressing COS cells is not inhibited by a monoclonal antibody (60.3) directed to an inhibitory epitope on CD18, indicating that the ELAM-1 ligand, although uncharacterized, is not a member of the CD11/CD18 family.