[Evaluation of the Clinical Outcomes of Autologous Chondrocyte Implantation in the Management of Chondral Lesions of the Knee after 10+ Years]. / Vyhodnocení klinických výsledku metody implantace solidního chondrograftu v lécbe chondrálních lézí kolenního kloubu po více nez 10 letech.

PURPOSE OF THE STUDY: The authors present the outcomes of more than ten-year clinical follow-up of patients who underwent surgical treatment of deep chondral defect of the knee (medial or lateral condyle). The method of treatment was the implantation of autologous cultured chondrocytes in the form of a solid chondral graft. The aim was also to compare the mid-term and long-term outcomes and to evaluate how the knee condition affects the everyday life and sports activities more than 10 years after surgery. MATERIAL AND METHODS Thirty patients of the total of 56 patients (26 patients dropped out of the long-term follow-up) operated in the period between 2001 and 2012 were available for retrospective evaluation of the clinical condition before surgery, at 1, 2, 5 years after surgery and at 10+ years after surgery. The mean follow-up period of patients was 14.5 years (10 - 20.5 years) after surgery. The clinical evaluation was performed using the Lysholm Knee Scoring Scale and the Tegner Activity Scale. RESULTS The mean preoperative Lysholm score of the followed-up study population was 37.5. During the fi rst two years, improvement was achieved to the maximum value of 83.1. At 5 years and subsequently also at more than 10 years after surgery, a slight decline was reported to the mean value of 78.6. When comparing the outcomes at 5 years and at more than 10 years after surgery, the decline in the value was statistically non-signifi cant. The mean value of the Tegner Activity Scale at more than 10 years after surgery was 4.5 points out of 10 points. DISCUSSION Management of a chondral defect especially in younger patients constitutes a common challenge in everyday orthopaedic practice. There are multiple methods at hand, all of which have their pros and cons. The size of the chondral lesion appears to be the limiting and decisive factor. The greatest pitfall are large chondral lesions (>4cm2 ), where many methods fail to yield satisfactory outcomes. One of the suitable options is the autologous chondrocyte implantation method. CONCLUSIONS The clinical outcomes of patients followed-up for more than 10 years after surgery do not show a statistically signifi cant decline compared to the mid-term outcomes. Based on the data obtained, we continue to consider the autologous chondrocyte implantation as an effective method to manage deep chondral defects in the knee. The patients were mostly able to get back to normal life, including their sports activities, with a signifi cant improvement of its quality compared to that before surgery. KEY WORDS: knee joint, chondrocytes, autologous cartilage implantation, long-term.

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.

Acetaldehyde at clinically relevant concentrations inhibits inward rectifier potassium current I(K1) in rat ventricular myocytes.

Considering the effects of alcohol on cardiac electrical behavior as well as the important role of the inward rectifier potassium current I(K1) in arrhythmogenesis, this study was aimed at the effect of acetaldehyde, the primary metabolite of ethanol, on I(K1) in rat ventricular myocytes. Acetaldehyde induced a reversible inhibition of I(K1) with IC(50) = 53.7+/-7.7 microM at -110 mV; a significant inhibition was documented even at clinically-relevant concentrations (at 3 microM by 13.1+/-3.0 %). The inhibition was voltage-independent at physiological voltages above -90 mV. The I(K1) changes under acetaldehyde may contribute to alcohol-induced alterations of cardiac electrophysiology, especially in individuals with a genetic defect of aldehyde dehydrogenase where the acetaldehyde level may be elevated.

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.

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.

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.

Propafenone blocks ATP-sensitive K+ channels in rabbit atrial and ventricular cardiomyocytes.

Propafenone, a class I antiarrhythmic agent, inhibits several membrane currents (I(Na), I(Ca), I(K), Ito), however, its effects on ATP-sensitive potassium current (I(K)ATP) of cardiac cells have not been tested. We evaluated the blocking effects of 0.1 to 100 microM propafenone applications at 35 degrees C on the whole-cell I(K)ATP as triggered by dinitrophenol (75 microM) in adult rabbit dissociated atrial and ventricular cardiomyocytes in comparison. The block of I(K)ATP by propafenone was dose-dependent, fully reversible and voltage-independent. The dose-response relation, as evaluated at 0 mV for atrial myocytes (ED50 = 1.26+/-0.17 microM, Hill number = 1.25+/-0.22) was significantly shifted to the left vs. that in ventricular myocytes (ED50 = 4.94+/-0.59 microM, Hill number = 1.22+/-0.14). It is concluded that propafenone blocks cardiac I(K)ATP at a single site with 4 times higher affinity for the drug in atrial myocytes. This block of cardiac I(K)ATP might play a role in the beneficial and adverse effects of the drug.

Control of cardiac performance by Ca-turnover.

A quantitative model of Ca-turnover in cardiac cells that incorporates negative feedback modulation of sarcolemmal calcium transport (via Ca channels and Na/Ca exchange) has been designed. The Na/Ca exchange current was expressed as INaCa = INaCar + delta INaCa. The component INaCar reflects slow changes of Ca2+ and Na+ concentrations and depends on the Na/K pump. delta INaCa is the fast component related to the Ca2+ transient. The single input to the model is an arbitrary sequence of intervals between excitations; outputs are sequences of calcium amounts transferred among the compartments during individual intervals. The model operates with a combination of discrete variables (amounts of Ca transferred during contraction, relaxation and rest) and continuous variables - slow changes in ionic concentrations. Since the model is not formalistic but respects the nature of the underlying elements of the system, it enables us to stimulate the known effects of cardiotropic drugs or to predict their unknown mechanisms by visualizing the changes in individual Ca compartments. By altering the parameters, the model also stimulates the known species and tissue differences in rate-dependent phenomena.

Use-dependent features of 4-aminopyridine block of transient outward current in rat ventricular myocytes.

The inhibition of the calcium-independent transient outward current (Ito) by the widely used blocker 4-aminopyridine (4-AP) was studied in adult Wistar rat ventricular myocytes, enzymatically isolated and voltage-clamped in the whole cell configuration using patch-clamp pipettes. 4-AP at 1 mmol/l concentration caused complete steady-state block of Ito at resting or hyperpolarized voltages. The block was partially removed during repeated depolarizations applied at frequencies above 0.25 Hz. Changes in the level of block during a depolarizing pulse (to + 40 mV) and during a following rest period (at -90 mV) were analyzed. On depolarization, the recovery from 4-AP block (at 0.5 mmol/l) started with a delay approximately corresponding to the time constant of Ito inactivation and then followed a monoexponential time course (time constant of about 44 ms). The restoration of block at a holding voltage of -90 mV, after recovery of Ito from inactivation, followed a monoexponential time course (time constant of about 1.2 s). The results are consistent with the hypothesis that the binding site for 4-AP at the Ito channel is available in the closed and open states but not when inactivated. Unblocking of Ito at stimulation intervals shorter than approximately 1 s may occur at 4-AP concentrations below 4 mmol/l.

A contraction-related component of slow inward current in dog ventricular muscle and its relation to Na(+)-Ca2+ exchange.

1. The slow inward current component related to contraction (Isic) was studied in voltage clamp experiments on canine ventricular trabeculae at 30 degrees C with the aims of (a) estimating its relation to electrogenic Na(+)-Ca2+ exchange and (b) comparing it with similar currents as reported in cardiac myocytes. 2. Isic may be recorded under conditions of augmented contractility in response to depolarizing pulses below the threshold of the classic slow inward current (presumably mediated by L-type Ca2+ channels). In responses to identical depolarizing clamp pulses the peak value of Isic is directly related to the amplitude of contraction (Fmax). Isic peaks about 60 ms after the onset of depolarization and declines with a half-time of about 110 ms. 3. The voltage threshold of Isic activation is the same as the threshold of contraction. The positive inotropic clamp preconditions shift both thresholds to more negative values of membrane voltage, i.e. below the threshold of the classic slow inward current. 4. Isic may also be recorded as a slowly decaying inwardly directed current 'tail' after depolarizing pulses. In this representation the peak value of Isic changes with duration of the depolarizing pulses, again in parallel with Fmax. In response to pulses shorter than 100 ms both variables increase with depolarization time. If initial conditions remain constant, further prolongation of the pulse does not significantly influence either one (tail currents follow a common envelope). 5. Isic differs from classic slow inward current by: (a) its direct relation to contraction, (b) the slower decay of the current tail on repolarization, (c) slower restitution corresponding to the mechanical restitution, (d) its relative insensitivity to Ca(2+)-blocking agents (the decrease of Isic is secondary to the negative inotropic of Ca(2+)-blocking agents (the decrease of Isic is secondary to the negative inotropic effect) and (e) its disappearance after Sr2+ substitution for Ca2+. 6. The manifestations of Isic in multicellular preparations do not differ significantly from those reported in isolated myocytes (in contrast to calcium current). 7. The analysis of the correlation between Isic and Fmax transients during trains of identical test depolarizing pulses at variable extra- and intracellular ionic concentrations (changes of [Ca2+]o, 50% Li+ substitution for Na+, strophanthidin) indicate that the observed effects conform to the predictions based on a quantitative model of Na(+)-Ca2+ exchange. 8. It is concluded that Isic is activated by a transient increase of [Ca2+]i, in consequence of the release from the reticular stores.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage dependence of force- and slow inward current restitution in ventricular muscle.

This study was aimed to assess the relationship among the voltage-dependent processes underlying the excitation-contraction coupling, viz. force restitution (FR), transmembrane Ca fluxes and Ca release. The experiments (n = 22) were performed on voltage-clamped dog trabeculae in which force and slow inward current were measured. Standard steady-state was achieved by clamp driving at 0.5 Hz, 300 ms, 70 mV depolarizing pulses from holding = resting potential at 30 degrees C. Voltage and duration of single pulses and intervals in between were varied according to five protocols. The voltage dependence of Ca release was tested by varying single pulses at equal steady-state, i.e., at equal Ca availability. Contractions could be elicited in absence of ICa (20-30 mV step) and in the presence of disproportionately small ICa (above 80 mV). The voltage dependence of Ca availability for the release was tested by constant test pulses following either a variable conditioning clamp pulse or a period of rest at a variable voltage. After a low voltage pulse and, hence, depressed or absent ICa, the test contraction is diminished in presence of normal or even augmented Isi at any test interval (i.e., FR is depressed). Diminished Ca influx thus reduces the Ca availability of the subsequent beat. During prolonged depolarization (by 60 mV and more) a tonic response appears, but a phasic response cannot be elicited (FR is inhibited). Upon subsequent repolarization FR starts from zero and is significantly enhanced. It is concluded that, during depolarization, Ca release channels are in an open state, thus allowing free recirculation of Ca, but no build-up of a sufficient Ca gradient at the release site.

Use-dependent effects of 4-aminopyridine on transient outward current in dog ventricular muscle.

The use-dependent features of 4-aminopyridine-induced block of the transient outward current were investigated under voltage clamp conditions in dog ventricular myocardial bundles. The block depended strongly on the pattern of voltage clamp pulses. Its level (settled in rested membranes) became deeper at early time during depolarization. In contrast, prolonged depolarization (above 50 to 100 ms) produced relief of block. Our results suggest that the block strongly depends on the state of the channel gating system.

4-Aminopyridine sensitive transient outward current in dog ventricular fibres.

(1) The depression of slow inward calcium current (ICa) induced by organic or inorganic inhibitors in voltage clamped dog ventricular preparations unmasks an early transient outward current (Ito). (2) Ito is depressed by 4-aminopyridine (1 mM) in a voltage dependent manner. (3) Ito appears in response to voltage steps above 40 mV (from holding voltage = resting voltage) and increases with raising the amplitude of clamp steps. (4) Within physiological range of membrane voltage Ito is smaller and decays several times faster than ICa. Time course of the decline is approximately exponential (tau = 25 +/- 6 ms at 80 mV above resting voltage). (5) Shifts of the holding voltage by 20 mV from the level of resting voltage alters the peak amplitude of Ito. It is increased by hyperpolarization and reduced by depolarization. (6) The recovery of Ito from inactivation at resting voltage was approximated by a single exponential. Time constant (390 ms) is about 15 times longer than the time constant of inactivation at 80 mV positive to the resting voltage.