The sarcolemmal Ca2+ efflux pathways, Na+-Ca2+-exchanger (NCX) and Ca2+-ATPase (PMCA), play a crucial role in the regulation of intracellular Ca2+ load and Ca2+ transient in cardiomyocytes. The distribution of these pathways between the t-tubular and surface membrane of ventricular cardiomyocytes varies between species and is not clear in human. Moreover, several studies suggest that this distribution changes during the development and heart diseases. However, the consequences of NCX and PMCA redistribution in human ventricular cardiomyocytes have not yet been elucidated. In this study, we aimed to address this point by using a mathematical model of the human ventricular myocyte incorporating t-tubules, dyadic spaces, and subsarcolemmal spaces. Effects of various combinations of t-tubular fractions of NCX and PMCA were explored, using values between 0.2 and 1 as reported in animal experiments under normal and pathological conditions. Small variations in the action potential duration (≤ 2%), but significant changes in the peak value of cytosolic Ca2+ transient (up to 17%) were observed at stimulation frequencies corresponding to the human heart rate at rest and during activity. The analysis of model results revealed that the changes in Ca2+ transient induced by redistribution of NCX and PMCA were mainly caused by alterations in Ca2+ concentrations in the subsarcolemmal spaces and cytosol during the diastolic phase of the stimulation cycle. The results suggest that redistribution of both transporters between the t-tubular and surface membranes contributes to changes in contractility in human ventricular cardiomyocytes during their development and heart disease and may promote arrhythmogenesis.
- MeSH
- akční potenciály MeSH
- biologické modely MeSH
- buněčná membrána metabolismus MeSH
- kardiomyocyty * metabolismus MeSH
- lidé MeSH
- modely kardiovaskulární MeSH
- pumpa pro výměnu sodíku a vápníku * metabolismus MeSH
- sarkolema * metabolismus MeSH
- srdeční komory * metabolismus MeSH
- vápník * metabolismus MeSH
- vápníková signalizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The transverse-axial tubular system (tubular system) of cardiomyocytes plays a key role in excitation-contraction coupling. To determine the area of the tubular membrane in relation to the area of the surface membrane, indirect measurements through the determination of membrane capacitances are currently used in addition to microscopic methods. Unlike existing electrophysiological methods based on an irreversible procedure (osmotic shock), the proposed new approach uses a reversible short-term intermittent increase in the electrical resistance of the extracellular medium. The resulting increase in the lumen resistance of the tubular system makes it possible to determine separate capacitances of the tubular and surface membranes. Based on the analysis of the time course of the capacitive current, computational relations were derived to quantify the elements of the electrical equivalent circuit of the measured cardiomyocyte including both capacitances. The exposition to isotonic low-conductivity sucrose solution is reversible which is the main advantage of the proposed approach allowing repetitive measurements on the same cell under control and sucrose solutions. Experiments on rat ventricular cardiomyocytes (n = 20) resulted in the surface and tubular capacitance values implying the fraction of tubular capacitance/area of 0.327 ± 0.018. We conclude that the newly proposed method provides results comparable to the data obtained by the currently used detubulation method and, in addition, by being reversible, allows repeated evaluation of surface and tubular membrane parameters on the same cell.
The transverse-axial tubular system (t-tubules) plays an essential role in excitation-contraction coupling in cardiomyocytes. Its remodelling is associated with various cardiac diseases. Numerous attempts were made to analyse characteristics essential for proper understanding of the t-tubules and their impact on cardiac cell function in health and disease. The currently available methodical approaches related to the fraction of the t-tubular membrane area produce diverse data. The widely used detubulation techniques cause irreversible cell impairment, thus, distinct cell samples have to be used for estimation of t-tubular parameters in untreated and detubulated cells. Our proposed alternative method is reversible and allows repetitive estimation of the fraction of t-tubular membrane (ft) in cardiomyocytes using short-term perfusion of the measured cell with a low-conductive isotonic sucrose solution. It results in a substantial increase in the electrical resistance of t-tubular lumen, thus, electrically separating the surface and t-tubular membranes. Using the whole-cell patch-clamp measurement and the new approach in enzymatically isolated rat atrial and ventricular myocytes, a set of data was measured and evaluated. The analysis of the electrical equivalent circuit resulted in the establishment of criteria for excluding measurements in which perfusion with a low conductivity solution did not affect the entire cell surface. As expected, the final average ft in ventricular myocytes (0.337 ± 0.017) was significantly higher than that in atrial myocytes (0.144 ± 0.015). The parameter ft could be estimated repetitively in a particular cell (0.345 ± 0.021 and 0.347 ± 0.023 in ventricular myocytes during the first and second sucrose perfusion, respectively). The new method is fast, simple, and leaves the measured cell intact. It can be applied in the course of experiments for which it is useful to estimate both the surface and t-tubular capacitance/area in a particular cell.
- Publikační typ
- časopisecké články MeSH
Bronchodilator aminophylline may induce atrial or less often ventricular arrhythmias. The mechanism of this proarrhythmic side effect has not been fully explained. Modifications of inward rectifier potassium (Kir) currents including IK1 are known to play an important role in arrhythmogenesis; however, no data on the aminophylline effect on these currents have been published. Hence, we tested the effect of aminophylline (3-100 μM) on IK1 in enzymatically isolated rat ventricular myocytes using the whole-cell patch-clamp technique. A dual steady-state effect of aminophylline was observed; either inhibition or activation was apparent in individual cells during the application of aminophylline at a given concentration. The smaller the magnitude of the control IK1, the more likely the activation of the current by aminophylline and vice versa. The effect was reversible; the relative changes at -50 and -110 mV did not differ. Using IK1 channel population model, the dual effect was explained by the interaction of aminophylline with two different channel populations, the first one being inhibited and the second one being activated. Considering various fractions of these two channel populations in individual cells, varying effects observed in the measured cells could be simulated. We propose that the dual aminophylline effect may be related to the direct and indirect effect of the drug on various Kir2.x subunits forming the homo- and heterotetrameric IK1 channels in a single cell. The observed IK1 changes induced by clinically relevant concentrations of aminophylline might contribute to arrhythmogenesis related to the use of this bronchodilator in clinical medicine.
- MeSH
- aminofylin škodlivé účinky MeSH
- bronchodilatancia škodlivé účinky MeSH
- draslík farmakologie MeSH
- draslíkové kanály dovnitř usměrňující * MeSH
- kardiomyocyty fyziologie MeSH
- krysa rodu rattus MeSH
- srdeční arytmie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The ratio between Na+-Ca2+ exchange current densities in t-tubular and surface membranes of rat ventricular cardiomyocytes (JNaCa-ratio) estimated from electrophysiological data published to date yields strikingly different values between 1.7 and nearly 40. Possible reasons for such divergence were analysed by Monte Carlo simulations assuming both normal and log-normal distribution of the measured data. The confidence intervals CI95 of the mean JNaCa-ratios computed from the reported data showed an overlap of values between 1 and 3, and between 0.3 and 4.3 in the case of normal and log-normal distribution, respectively. Further analyses revealed that the published high values likely result from a large scatter of data due to transmural differences in JNaCa, dispersion of cell membrane capacitances and variability in incomplete detubulation. Taking into account the asymmetric distribution of the measured data, the reduction of mean current densities after detubulation and the substantially smaller CI95 of lower values of the mean JNaCa-ratio, the values between 1.6 and 3.2 may be considered as the most accurate estimates. This implies that 40 to 60% of Na+-Ca2+ exchanger is located at the t-tubular membrane of adult rat ventricular cardiomyocytes.
- MeSH
- kardiomyocyty * metabolismus MeSH
- krysa rodu rattus MeSH
- pumpa pro výměnu sodíku a vápníku MeSH
- sarkolema metabolismus MeSH
- sodík metabolismus MeSH
- srdeční komory metabolismus MeSH
- vápník * metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The current density (J) is a parameter routinely used to characterize individual ionic membrane currents. Its evaluation is based on the presumption that the magnitude of whole-cell ionic membrane current (I) is directly proportional to the cell membrane capacitance (C), i.e. I positively and strongly correlates with C and the regression line describing I-C relation intersects the y-axis close to the origin of coordinates. We aimed to prove the presumption in several examples and find whether the conversion of I to J could be always beneficial. I-C relation was analysed in several potassium currents, measured in rat atrial myocytes (in inward rectifier currents, IK1, and both the constitutively active and acetylcholine-induced components of acetylcholine-sensitive current, IK(Ach)CONST and IK(Ach)ACH), and in rat ventricular myocytes (transient outward current Ito). I-C correlation was estimated by the Pearson coefficient (r). A coefficient (k) was newly suggested describing deviation of the regression intercept from zero in currents with considerable r value. Based on mathematical simulations, I was satisfactorily proportional to C when r ≥ 0.6 and k ≤ 0.2 which was fulfilled in IK1 and IK(Ach)ACH (r = 0.84, k = 0.20, and r = 0.61, k = 0.06, respectively). I-C correlation was significantly positive, but weak in IK(Ach)CONST (r = 0.42), and virtually missing in Ito (r = 0.04). The impaired I-C proportionality in IK(Ach)CONST and Ito likely reflects heterogeneity of the channel expression. We conclude that the conversion of I to J should be avoided when I-C proportionality is absent. Otherwise, serious misinterpretation of data may arise.
- MeSH
- acetylcholin chemie MeSH
- buněčná membrána fyziologie MeSH
- elektrická kapacitance MeSH
- elektrofyziologie MeSH
- genotyp MeSH
- ionty MeSH
- kardiomyocyty účinky léků MeSH
- krysa rodu rattus MeSH
- membránové potenciály účinky léků MeSH
- myokard MeSH
- potkani Wistar MeSH
- srdeční síně patologie MeSH
- svalové buňky cytologie MeSH
- teoretické modely MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The distribution of data presented in many electrophysiological studies is presumed to be normal without any convincing evidence. To test this presumption, the cell membrane capacitance and magnitude of inward rectifier potassium currents were recorded by the whole-cell patch clamp technique in rat atrial myocytes. Statistical analysis of the data showed that these variables were not distributed normally. Instead, a positively skewed distribution appeared to be a better approximation of the real data distribution. Consequently, the arithmetic mean, used inappropriately in such data, may substantially overestimate the true mean value characterizing the central tendency of the data. Moreover, a large standard deviation describing the variance of positively skewed data allowed 95% confidence interval to include unrealistic negative values. We therefore conclude that the normality of the electrophysiological data should be tested in every experiment and, if rejected, the positively skewed data should be more accurately characterized by the median and interpercentile range or, if justified (namely in the case of log-normal and gamma data distribution), by the geometric mean and the geometric standard deviation.
- MeSH
- algoritmy MeSH
- buněčná membrána patologie fyziologie MeSH
- elektrická kapacitance MeSH
- elektrody MeSH
- elektrofyziologie metody MeSH
- interpretace statistických dat MeSH
- krysa rodu rattus MeSH
- membránové potenciály MeSH
- normální rozdělení * MeSH
- potkani Wistar MeSH
- reprodukovatelnost výsledků MeSH
- srdeční síně patologie MeSH
- svalové buňky fyziologie MeSH
- teoretické modely MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A variety of techniques of cell capacitance measurement have been proposed and applied in cellular electrophysiology. They are mostly based on the evaluation of membrane current responses to small changes in the membrane voltage. One of the currently used approaches applies the least-squares fit of an exponential current decay in response to voltage clamped rectangular pulses. In this study, we propose an alternative simpler approach to evaluation of the exponential parts in the current responses to square wave stimulation and present preliminary results of membrane capacitance evaluation. It is based on the property of the exponential function that has not yet been used to measure membrane capacitance. The time constant and the asymptote of the exponential waveform are unambiguously determined by the values read at three points separated by a constant time interval. In order to minimize the effect of noise and deviations from the exponential waveform, the triplet of points is designed to slide along the time axis. The results of the proposed approach and those previously evaluated by the least squares method are comparable. The method described may be advantageous for continuously recording changes in membrane capacitance.
- MeSH
- algoritmy MeSH
- elektrická kapacitance MeSH
- elektrická vodivost MeSH
- elektrofyziologie MeSH
- krysa rodu rattus MeSH
- membránové potenciály fyziologie MeSH
- metoda nejmenších čtverců MeSH
- metoda terčíkového zámku metody MeSH
- modely neurologické MeSH
- neurony MeSH
- počítačová simulace MeSH
- reprodukovatelnost výsledků MeSH
- software MeSH
- srdeční síně patologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Recent experimental work has revealed unusual features of the effect of certain drugs on cardiac inwardly rectifying potassium currents, including the constitutively active and acetylcholine-induced components of acetylcholine-sensitive current (IKAch). These unusual features have included alternating susceptibility of the current components to activation and inhibition induced by ethanol or nicotine applied at various concentrations, and significant correlation between the drug effect and the current magnitude measured under drug-free conditions. To explain these complex drug effects, we have developed a new type of quantitative model to offer a possible interpretation of the effect of ethanol and nicotine on the IKAch channels. The model is based on a description of IKAch as a sum of particular currents related to the populations of channels formed by identical assemblies of different α-subunits. Assuming two different channel populations in agreement with the two reported functional IKAch-channels (GIRK1/4 and GIRK4), the model was able to simulate all the above-mentioned characteristic features of drug-channel interactions and also the dispersion of the current measured in different cells. The formulation of our model equations allows the model to be incorporated easily into the existing integrative models of electrical activity of cardiac cells involving quantitative description of IKAch. We suppose that the model could also help make sense of certain observations related to the channels that do not show inward rectification. This new ionic channel model, based on a concept we call population type, may allow for the interpretation of complex interactions of drugs with ionic channels of various types, which cannot be done using the ionic channel models available so far.
- MeSH
- acetylcholin farmakologie MeSH
- biologické modely MeSH
- časové faktory MeSH
- dovnitř usměrňující draslíkové kanály spřažené s G proteiny metabolismus MeSH
- ethanol farmakologie MeSH
- gating iontového kanálu účinky léků MeSH
- nikotin farmakologie MeSH
- počítačová simulace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Inward rectifier potassium currents (I Kir,x) belong to prominent ionic currents affecting both resting membrane voltage and action potential repolarization in cardiomyocytes. In existing integrative models of electrical activity of cardiac cells, they have been described as single current components. The proposed quantitative model complies with findings indicating that these channels are formed by various homomeric or heteromeric assemblies of channel subunits with specific functional properties. Each I Kir,x may be expressed as a total of independent currents via individual populations of identical channels, i.e., channels formed by the same combination of their subunits. Solution of the model equations simulated well recently observed unique manifestations of dual ethanol effect in rat ventricular and atrial cells. The model reflects reported occurrence of at least two binding sites for ethanol within I Kir,x channels related to slow allosteric conformation changes governing channel conductance and inducing current activation or inhibition. Our new model may considerably improve the existing models of cardiac cells by including the model equations proposed here in the particular case of the voltage-independent drug-channel interaction. Such improved integrative models may provide more precise and, thus, more physiologically relevant results.
- MeSH
- akční potenciály * MeSH
- alosterická regulace MeSH
- draslíkové kanály dovnitř usměrňující chemie metabolismus MeSH
- ethanol farmakologie MeSH
- kardiomyocyty účinky léků metabolismus fyziologie MeSH
- krysa rodu rattus MeSH
- modely kardiovaskulární MeSH
- multimerizace proteinu MeSH
- srdce - funkce komor MeSH
- srdeční komory cytologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
