92 results on '"Zahradník I"'
Search Results
2. Analysis of noisy transient signals based on Gaussian process regression
- Author
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Baglaeva, I., primary, Iaparov, B., additional, Zahradník, I., additional, and Zahradníková, A., additional
- Published
- 2022
- Full Text
- View/download PDF
3. Ryanodine Receptor Adaptation
- Author
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Fill, M., Zahradníková, A., Villalba-Galea, C. A., Zahradník, I., Escobar, A. L., and Györke, S.
- Published
- 2000
4. Estimation of passive electrical parameters of living cells using a voltage step stimulus
- Author
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Novák, P., Marko, M., and Zahradník, I.
- Published
- 2000
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5. Recovery of calcium current from release-dependent inactivation in cardiac myocytes
- Author
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Kubalova, Z., Pavelkova, J., Zahradníková, A., and Zahradník, I.
- Published
- 2000
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6. Presence of several fast-access closed states in steady-state ryanodine receptor activity
- Author
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Dura, M., Zahradník, I., Györke, S., and Zahradníková, A.
- Published
- 2000
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7. Rapid Activation of the Cardiac Ryanodine Receptor by Submillisecond Calcium Stimuli
- Author
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Zahradníková, A., Zahradník, I., Györke, I., and Györke, S.
- Published
- 1999
8. Analysis of the inner and outer environment of sarcolemma in cardiac muscle cells. Nichtová Z.1,2, Novotová M.1, Zahradník I1
- Author
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Nichtova, Zuzana, Novotová, M., and Zahradník, I.
- Published
- 2014
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9. SUBŠTRUKTÚRA A VNÚTROBUNKOVÉ PROSTREDIE INTERKALÁRNYCH DISKOV SRDCOVÝCH SVALOVÝCH BUNIEK Nichtová Z (1, 2), Novotová M (1), Zahradník I (1)
- Author
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Nichtova, Zuzana, Novotová, M., and Zahradník, I.
- Published
- 2014
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10. DOMINANT MICRODOMAINS OF PLASMA MEMBRANE OF CARDIAC MYOCYTES Zuzana Nichtová1, 2, Marta Novotová1, Ivan Zahradník1
- Author
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Nichtova, Zuzana, Novotová, M., and Zahradník, I.
- Published
- 2014
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11. Changes in dyad morphogenesis in cardiac myocytes due to isoproterenol-induced myocardial injury Novotová M1, Nichtová Z1,2, Zahradník I.1
- Author
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Novotová, M. and Nichtová Z. Zahradník, I.
- Published
- 2013
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12. Inner and outer environment of plasma membrane in cardiac myocytes Nichtová Z.1,2, Zahradník I.1, Novotová M.1
- Author
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Nichtova, Zuzana, Zahradník I, and Novotová M
- Published
- 2013
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13. Implementation of the method of correlative light/electron microscopy on the same cell J. Sukeľová1, Z. Nichtová1,2, M. Novotová1, I. Zahradník1
- Author
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Súkeľová, J., Nichtová, Z., Novotová, M., and Zahradník, I.
- Published
- 2012
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14. The plasticity of the plasma membrane of cardiac myocytes Zuzana Nichtová1, 2, Ivan Zahradník1, Tatiana Stankovičová2, Marta Novotová1
- Author
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Nichtova, Zuzana, Zahradník, I., Stankovičová, T., and Novotová, M.
- Published
- 2012
- Full Text
- View/download PDF
15. Regulation of dynamic behavior of cardiac ryanodine receptor by Mg2+under simulated physiological conditions
- Author
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Zahradníková, A., primary, Dura, M., additional, Györke, I., additional, Escobar, A. L., additional, Zahradník, I., additional, and Györke, S., additional
- Published
- 2003
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16. Kinetics of cardiac RyR channel gating studied at high temporal resolution.
- Author
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Dura, M, primary, Zahradník, I, additional, and Zahradníková, A, additional
- Published
- 2003
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17. Effect of the High-Fat Diet on the Calcium Channels in Rat Myocardium
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MINAROVIČ, I., primary, VOJTKO, R., additional, ŠEBÖKOVÁ, E., additional, KLIMEŠ, I., additional, and ZAHRADNÍK, I., additional
- Published
- 1997
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18. A minimal gating model for the cardiac calcium release channel
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Zahradníková, A., primary and Zahradník, I., additional
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- 1996
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19. Description of modal gating of the cardiac calcium release channel in planar lipid membranes
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Zahradníková, A., primary and Zahradník, I., additional
- Published
- 1995
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20. Regulation of dynamic behavior of cardiac ryanodine receptor by Mg[sup 2+] under simulated physiological conditions.
- Author
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Zahradníková, A., Dura, M., Györke, I., Escobar, A.L., Zahradník, I., and Györke, S.
- Subjects
MUSCLE cells ,MYOCARDIUM ,MAGNESIUM ,CALCIUM ,RYANODINE - Abstract
Mg[sup 2+], an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca[sup 2+] release channels by competing with Ca[sup 2+] at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca[sup 2+]-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca[sup 2+] stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca[sup 2+] compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg[sup 2+] at the background of 3 mM total ATP in the presence or absence of i mM luminal Ca[sup 2+]. Mg[sup 2+] dramatically slowed the rate of activation of RyRs in response to sustained (≥10-ms) elevations in Ca[sup 2+] concentration. Paradoxically, Mg[sup 2+] had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (<1-ms) Ca[sup 2+] stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca[sup 2+] did not affect the peak open probability or the probability of responses to brief Ca[sup 2+] signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg[sup 2+] is a critical physiological determinant of the dynamic behavior of the RyR channel, which is expected to profoundly influence the fidelity of coupling between L-type Ca[sup 2+] channels and RyRs in heart cells. [ABSTRACT FROM AUTHOR]
- Published
- 2003
21. Effect of the High-Fat Diet on the Calcium Channels in Rat Myocardiuma.
- Author
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MINAROVIČ, I., VOJTKO, R., ŠEBÖKOVÁ, E., KLIMEŠ, I., and ZAHRADNÍK, I.
- Published
- 1997
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22. Effect of the High-Fat Diet on the Calcium Channels in Rat Myocardium a
- Author
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MINAROVI, I., VOJTKO, R., SEBÖKOVÁ, E., KLIMES, I., and ZAHRADNÍK, I.
- Published
- 1997
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23. Optimization of a neutron dosimeter for the high energy accelerators
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Sokolov Alexey, Fehrenbacher Georg, Kozlova Ekaterina, Radon Torsten, and Zahradnik Izabella
- Subjects
Physics ,QC1-999 - Abstract
In high energy accelerator facilities the neutron radiation should be continuously measured during operation to control the ambient dose. This requires a reliable neutron dosimeter in a wide energy range. In this work we present an optimization of a compact cylindrical passive neutron dosimeter for the usage in wide energy neutron fields.
- Published
- 2017
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24. Analysis of noisy transient signals based on Gaussian process regression.
- Author
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Baglaeva I, Iaparov B, Zahradník I, and Zahradníková A
- Subjects
- Signal-To-Noise Ratio, Software, Algorithms
- Abstract
Dynamic systems such as cells or tissues generate, either spontaneously or in response to stimuli, transient signals that carry information about the system. Characterization of recorded transients is often hampered by a low signal-to-noise ratio (SNR). Reduction of the noise by filtering has limited use due to partial signal distortion. Occasionally, transients can be approximated by a mathematical function, but such a function may not hold correctly if recording conditions change. We introduce here the model-independent approximation method for general noisy transient signals based on the Gaussian process regression. The method was implemented in the software TransientAnalyzer, which detects transients in a record, finds their best approximation by the Gaussian process, constructs a surrogate spline function, and estimates specified signal parameters. The method and software were tested on a cellular model of the calcium concentration transient corrupted by various SNR levels and recorded at a low sampling frequency. Statistical analysis of the model data sets provided the error of estimation <7.5% and the coefficient of variation of estimates <17% for peak SNR = 5. The performance of Gaussian process regression on signals of diverse experimental origin was even better than fitting by a function. The software and its description are available on GitHub., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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25. SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells.
- Author
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Koči J, Novotová M, Sláviková M, Klempa B, and Zahradník I
- Abstract
The coronavirus transforms the cytoplasm of susceptible cells to support virus replication. It also activates autophagy-like processes, the role of which is not well understood. Here, we studied SARS-CoV-2-infected Vero E6 cells using transmission electron microscopy and autophagy PCR array. After 6-24 h post-infection (hpi), the cytoplasm of infected cells only contained double-membrane vesicles, phagophores, and phagosomes engulfing virus particles and cytoplasmic debris, including damaged mitochondria. The phagosomes interacted with the viral nucleoprotein complex, virus particles, mitochondria, and lipid droplets. The phagosomes transformed into egress vacuoles, which broke through the plasmalemma and discharged the virus particles. The Vero E6 cells exhibited pronounced virus replication at 6 hpi, which stabilized at 18-24 hpi at a high level. The autophagy PCR array tests revealed a significant upregulation of 10 and downregulation of 8 autophagic gene markers out of 84. Altogether, these results underline the importance of autophagy-like processes for SARS-CoV-2 maturation and egress, and point to deviations from a canonical autophagy response.
- Published
- 2022
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26. Magnesium Ions Moderate Calcium-Induced Calcium Release in Cardiac Calcium Release Sites by Binding to Ryanodine Receptor Activation and Inhibition Sites.
- Author
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Iaparov B, Baglaeva I, Zahradník I, and Zahradníková A
- Abstract
Ryanodine receptor channels at calcium release sites of cardiac myocytes operate on the principle of calcium-induced calcium release. In vitro experiments revealed competition of Ca
2+ and Mg2+ in the activation of ryanodine receptors (RyRs) as well as inhibition of RyRs by Mg2+ . The impact of RyR modulation by Mg2+ on calcium release is not well understood due to the technical limitations of in situ experiments. We turned instead to an in silico model of a calcium release site (CRS), based on a homotetrameric model of RyR gating with kinetic parameters determined from in vitro measurements. We inspected changes in the activity of the CRS model in response to a random opening of one of 20 realistically distributed RyRs, arising from Ca2+ /Mg2+ interactions at RyR channels. Calcium release events (CREs) were simulated at a range of Mg2+ -binding parameters at near-physiological Mg2+ and ATP concentrations. Facilitation of Mg2+ binding to the RyR activation site inhibited the formation of sparks and slowed down their activation. Impeding Mg-binding to the RyR activation site enhanced spark formation and speeded up their activation. Varying Mg2+ binding to the RyR inhibition site also dramatically affected calcium release events. Facilitation of Mg2+ binding to the RyR inhibition site reduced the amplitude, relative occurrence, and the time-to-end of sparks, and vice versa. The characteristics of CREs correlated dose-dependently with the effective coupling strength between RyRs, defined as a function of RyR vicinity, single-channel calcium current, and Mg-binding parameters of the RyR channels. These findings postulate the role of Mg2+ in calcium release as a negative modulator of the coupling strength among RyRs in a CRS, translating to damping of the positive feedback of the calcium-induced calcium-release mechanism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Iaparov, Baglaeva, Zahradník and Zahradníková.)- Published
- 2022
- Full Text
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27. Toward more accurate data in cardiac cellular electrophysiology.
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Bébarová M, Pásek M, and Zahradník I
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- Animals, Calcium metabolism, Cell Membrane physiology, Humans, Myocytes, Cardiac physiology, Reproducibility of Results, Cardiology standards, Electrophysiology standards
- Published
- 2020
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28. The problem of accuracy in single-channel open probability measurements.
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Zahradníková A, Iaparov B, and Zahradník I
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- Animals, Humans, Kinetics, Membrane Potentials, Myocytes, Cardiac cytology, Patch-Clamp Techniques, Probability, Reproducibility of Results, Electrophysiology methods, Ion Channel Gating, Ion Channels physiology
- Abstract
The function of ion channels to mediate the flux of ions through membranes of living cells depends on their number, conductance, and open probability. The open probability, P
O , characterizes gating of channels that is sensitive to experimental conditions and that can be determined in single-channel experiments. Individual experimental records and even whole series of single-channel activity measurements represent random samples of the stochastic gating continuous in time. The aim of this study was to understand the relationship between the accuracy (trueness and precision) of PO determination and the method of single-channel activity data collection. We used simulated single-channel experiments with variable settings of data collection for a range of open probability values. We found that at low PO , the trueness of PO determination depends on the average number of channel openings per record, while the precision of PO determination depends on the total number of channel openings in the whole dataset and on the distribution of open and closed times. We derived relationships that allow planning of single-channel experiments for the required accuracy of PO determination over a large span of open probabilities., Competing Interests: Declarations of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)- Published
- 2020
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29. Structural variability of dyads relates to calcium release in rat ventricular myocytes.
- Author
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Novotová M, Zahradníková A Jr, Nichtová Z, Kováč R, Kráľová E, Stankovičová T, Zahradníková A, and Zahradník I
- Subjects
- Animals, Excitation Contraction Coupling, Male, Myocytes, Cardiac cytology, Rats, Rats, Wistar, Sarcoplasmic Reticulum metabolism, Calcium metabolism, Calcium Channels metabolism, Calcium Signaling, Heart Ventricles physiopathology, Myocardial Contraction, Myocytes, Cardiac physiology
- Abstract
Cardiac excitation-contraction coupling relies on dyads, the intracellular calcium synapses of cardiac myocytes, where the plasma membrane contacts sarcoplasmic reticulum and where electrical excitation triggers calcium release. The morphology of dyads and dynamics of local calcium release vary substantially. To better understand the correspondence between the structure and the functionality of dyads, we estimated incidences of structurally different dyads and of kinetically different calcium release sites and tested their responsiveness to experimental myocardial injury in left ventricular myocytes of rats. According to the structure of dyads estimated in random electron microscopic images of myocardial tissue, the dyads were sorted into 'compact' or 'loose' types. The calcium release fluxes, triggered at local calcium release sites in patch-clamped ventricular myocytes and recorded by laser scanning confocal fluorescence microscopy, were decomposed into 'early' and 'late' components. ANOVA tests revealed very high correlation between the relative amplitudes of early and late calcium release flux components and the relative occurrences of compact and loose dyads in the control and in the injured myocardium. This finding ascertained the relationship between the structure of dyads and the functionality of calcium release sites and the responsiveness of calcium release sites to physical load in cardiac myocytes.
- Published
- 2020
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30. Calcium Signaling and Contractility in Cardiac Myocyte of Wolframin Deficient Rats.
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Cagalinec M, Zahradníková A, Zahradníková A Jr, Kováčová D, Paulis L, Kureková S, Hot'ka M, Pavelková J, Plaas M, Novotová M, and Zahradník I
- Abstract
Wolframin (Wfs1) is a membrane protein of the sarco/endoplasmic reticulum. Wfs1 mutations are responsible for the Wolfram syndrome, characterized by diabetic and neurological symptoms. Although Wfs1 is expressed in cardiac muscle, its role in this tissue is not clear. We have characterized the effect of invalidation of Wfs1 on calcium signaling-related processes in isolated ventricular myocytes of exon5-Wfs1 deficient rats (Wfs1
-e5/-e5 ) before the onset of overt disease. Calcium transients and contraction were measured in field-stimulated isolated myocytes using confocal microscopy with calcium indicator fluo-3 AM and sarcomere length detection. Calcium currents and their calcium release-dependent inactivation were characterized in whole-cell patch-clamp experiments. At 4 months, Wfs1-e5/-e5 animals were euglycemic, and echocardiographic examination revealed fully compensated cardiac function. In field-stimulated isolated ventricular myocytes, both the amplitude and the duration of contraction of Wfs1-e5/-e5 animals were elevated relative to control Wfs1+/+ littermates. Increased contractility of myocytes resulted largely from prolonged cytosolic calcium transients. Neither the amplitude of calcium currents nor their voltage dependence of activation differed between the two groups. Calcium currents in Wfs1-e5/-e5 myocytes showed a larger extent of inactivation by short voltage prepulses applied to selectively induce calcium release-dependent inactivation of calcium current. Neither the calcium content of the sarcoplasmic reticulum, measured by application of 20 mmol/l caffeine, nor the expression of SERCA2, determined from Western blots, differed significantly in myocytes of Wfs1-e5/-e5 animals compared to control ones. These experiments point to increased duration of calcium release in ventricular myocytes of Wfs1-e5/-e5 animals. We speculate that the lack of functional wolframin might cause changes leading to upregulation of RyR2 channels resulting in prolongation of channel openings and/or a delay in termination of calcium release.- Published
- 2019
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31. Calcium release-dependent inactivation precedes formation of the tubular system in developing rat cardiac myocytes.
- Author
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Macková K, Zahradníková A Jr, Hoťka M, Hoffmannová B, Zahradník I, and Zahradníková A
- Subjects
- Animals, Calcium Channels metabolism, Cell Proliferation, Electrophysiological Phenomena, Female, Male, Rats, Calcium metabolism, Myocytes, Cardiac cytology, Myocytes, Cardiac metabolism
- Abstract
Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.
- Published
- 2017
- Full Text
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32. Reconstruction of membrane current by deconvolution and its application to membrane capacitance measurements in cardiac myocytes.
- Author
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Hoťka M and Zahradník I
- Subjects
- Animals, Cell Separation, Male, Membrane Fusion, Microelectrodes, Rats, Cell Membrane physiology, Electric Capacitance, Membrane Potentials physiology, Myocytes, Cardiac physiology
- Abstract
Correct detection of membrane currents under whole-cell patch-clamp conditions is limited by the transfer function of a recording system. The low-pass output filter of a recording amplifier alters the time course of membrane current and causes errors in relevant descriptors. To solve these problems, we developed a practical procedure for reconstruction of the time course of membrane currents based on deconvolution of recorded currents in frequency domain. The procedure was tested on membrane capacitance estimates from current responses to step voltage pulses. The reconstructed current responses, in contrast to original current records, could be described exactly by an adequate impedance model of a recorded cell. The reconstruction allowed to increase the accuracy and reliability of membrane capacitance measurements in wide range of cell sizes and to suppress the cross-talk errors well below the noise. Moreover, it allowed resolving the instabilities in recording conditions arising from parasitic capacitance and seal resistance variation. Complex tests on hardware models, on simulated data sets, and on living cells confirmed robustness and reliability of the deconvolution procedure. The aptitude of the method was demonstrated in isolated rat cardiac myocytes by recording of spontaneous vesicular events, by discerning the formation of a fusion pore, and by revealing artefacts due to unstable seal resistance.
- Published
- 2017
- Full Text
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33. Ultrastructural remodelling of slow skeletal muscle fibres in creatine kinase deficient mice: a quantitative study.
- Author
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Novotová M, Tarabová B, Tylková L, Ventura-Clapier R, and Zahradník I
- Subjects
- Animals, Cells, Cultured, Creatine Kinase metabolism, Male, Mice, Mice, Inbred C57BL, Mitochondria, Muscle enzymology, Mitochondria, Muscle pathology, Muscle Fibers, Slow-Twitch pathology, Sarcoplasmic Reticulum pathology, Creatine Kinase deficiency, Mitochondria, Muscle ultrastructure, Muscle Fibers, Slow-Twitch enzymology, Muscle Fibers, Slow-Twitch ultrastructure, Sarcoplasmic Reticulum enzymology, Sarcoplasmic Reticulum ultrastructure
- Abstract
Creatine kinase content, isoform distribution, and participation in energy transfer are muscle type specific. We analysed ultrastructural changes in slow muscle fibres of soleus due to invalidation of creatine kinase (CK) to reveal a difference in the remodelling strategy in comparison with fast muscle fibres of gastrocnemius published previously. We have employed the stereological method of vertical sections and electron microscopy of soleus muscles of wild type (WT) and CK-/- mice. The mitochondrial volume density was 1.4× higher but that of sarcoplasmic reticulum (SR) was almost 5× lower in slow CK-/- muscles fibres than in WT fibres. The volume density of terminal cisterns and of t-tubules was also lower in CK-/- than in WT fibres. The analysis of organelle environment revealed increased neighbourhood of mitochondria and A-bands that resulted from the decreased volume density of SR, from relocation of mitochondria along myofibrils, and from intrusion of mitochondria to myofibrils. These processes direct ATP supply closer to the contractile machinery. The decreased interaction between mitochondria and SR suggests reduced dependence of calcium uptake on oxidative ATP production. In conclusion, the architecture of skeletal muscle cells is under control of a cellular program that optimizes energy utilization specifically for a given muscle type.
- Published
- 2016
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34. Quantitative analysis of calcium spikes in noisy fluorescent background.
- Author
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Janicek R, Hotka M, Zahradníková A Jr, Zahradníková A, and Zahradník I
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- Algorithms, Animals, Calcium metabolism, Fluorescent Dyes, Microscopy, Confocal, Microscopy, Fluorescence, Myocytes, Cardiac cytology, Patch-Clamp Techniques, Rats, Ryanodine Receptor Calcium Release Channel analysis, Ryanodine Receptor Calcium Release Channel metabolism, Signal-To-Noise Ratio, Calcium analysis, Calcium Signaling, Models, Statistical, Myocytes, Cardiac metabolism, Software
- Abstract
Intracellular calcium signals are studied by laser-scanning confocal fluorescence microscopy. The required spatio-temporal resolution makes description of calcium signals difficult because of the low signal-to-noise ratio. We designed a new procedure of calcium spike analysis based on their fitting with a model. The accuracy and precision of calcium spike description were tested on synthetic datasets generated either with randomly varied spike parameters and Gaussian noise of constant amplitude, or with constant spike parameters and Gaussian noise of various amplitudes. Statistical analysis was used to evaluate the performance of spike fitting algorithms. The procedure was optimized for reliable estimation of calcium spike parameters and for dismissal of false events. A new algorithm was introduced that corrects the acquisition time of pixels in line-scan images that is in error due to sequential acquisition of individual pixels along the space coordinate. New software was developed in Matlab and provided for general use. It allows interactive dissection of temporal profiles of calcium spikes from x-t images, their fitting with predefined function(s) and acceptance of results on statistical grounds, thus allowing efficient analysis and reliable description of calcium signaling in cardiac myocytes down to the in situ function of ryanodine receptors.
- Published
- 2013
- Full Text
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35. Calcium spike variability in cardiac myocytes results from activation of small cohorts of ryanodine receptor 2 channels.
- Author
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Janiek R, Zahradníková A Jr, Poláková E, Pavelková J, Zahradník I, and Zahradníková A
- Subjects
- Animals, In Vitro Techniques, Ion Channel Gating, Male, Myocytes, Cardiac physiology, Rats, Rats, Wistar, Calcium physiology, Calcium Signaling physiology, Ryanodine Receptor Calcium Release Channel physiology
- Abstract
In mammalian cardiac myocytes, the elementary calcium releases triggered by step voltage stimuli manifest either as solitary or as twin spikes that vary widely in kinetics and amplitude for unknown reasons. Here we examined the variability of calcium spikes measured using line-scanning confocal microscopy in patch-clamped rat ventricular myocytes. Amplitude distributions of the single and of the first of twin spikes were broader than those of the second spikes. All could be best approximated by a sum of a few elementary Gaussian probability distribution functions. The latency distributions of the single and the first spikes were identical, much shorter and less variable than those of the second spikes. The multimodal distribution of spike amplitudes and the probability of occurrence of twin spikes were stochastically congruent with activation of only a few of the many RyR2 channels present in the release site cluster. The occurrence of twin release events was rare due to refractoriness of release, induced with a probability proportional to the number of RyR2s activated in the primary release event. We conclude that the variability of the elementary calcium release events supports a calcium signalling mechanism that arises from stochastics of RyR2 gating and from inactivation of local origin.
- Published
- 2012
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36. Construction of calcium release sites in cardiac myocytes.
- Author
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Zahradníková A and Zahradník I
- Abstract
Local character of calcium release in cardiac myocytes, as defined by confocal recordings of calcium sparks, implies independent activation of individual calcium release sites based on ryanodine receptor (RyR) channel recruitment. We constructed virtual calcium release sites (vCRSs) composed of a variable number of RyR channels distributed in clusters in accordance with the experimentally observed cluster size distribution. The vCRSs consisted either of a single virtual calcium release unit (vCRU), in which all clusters shared a common dyadic space, or of multiple virtual calcium release units (CRUs) containing one cluster each and having separate dyadic spaces. We explored the stochastic behavior of vCRSs to understand the activation and recruitment of RyRs during calcium sparks. RyRs were represented by the published allosteric gating model that included regulation by cytosolic Ca(2+) and Mg(2+). The interaction of Mg(2+) with the RyR Ca(2+)-binding sites and the refractory period of vCRSs were optimized to accord with the experimentally observed calcium dependence of calcium spark frequency. The Mg(2+)-binding parameters of RyRs that provided the best description of spark frequency depended on the number of RyRs assembled in the vCRSs. Adequate inhibitory effect of Mg(2+) on the calcium dependence of RyR open probability was achieved if the vCRSs contained at least three clusters. For the distribution of the number of open RyRs in evoked calcium sparks to correspond to the experimentally observed distribution of spark calcium release fluxes, at least three clusters had to share a common virtual CRU, in which ∼3 RyRs open to form an average spark. These results reconcile the small cluster size and stochastic placement of RyRs in the release sites with the estimates of the amount of RyR protein, volume density of calcium release sites, and the size of calcium release sites in rat cardiac myocytes.
- Published
- 2012
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37. Challenging quantal calcium signaling in cardiac myocytes.
- Author
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Zahradníková A, Gaburjáková M, Bridge JH, and Zahradník I
- Subjects
- Animals, Models, Theoretical, Myocytes, Cardiac cytology, Rats, Ryanodine Receptor Calcium Release Channel physiology, Calcium Signaling physiology, Myocytes, Cardiac physiology
- Published
- 2010
- Full Text
- View/download PDF
38. Frequency and release flux of calcium sparks in rat cardiac myocytes: a relation to RYR gating.
- Author
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Zahradníková A, Valent I, and Zahradník I
- Subjects
- Algorithms, Allosteric Regulation physiology, Animals, Calcium metabolism, Calcium Channels, L-Type physiology, Computer Simulation, Diastole physiology, Kinetics, Magnesium metabolism, Models, Molecular, Rats, Ryanodine Receptor Calcium Release Channel chemistry, Systole physiology, Calcium Signaling physiology, Ion Channel Gating physiology, Models, Cardiovascular, Myocytes, Cardiac physiology, Ryanodine Receptor Calcium Release Channel physiology
- Abstract
Cytosolic calcium concentration in resting cardiac myocytes locally fluctuates as a result of spontaneous microscopic Ca(2+) releases or abruptly rises as a result of an external trigger. These processes, observed as calcium sparks, are fundamental for proper function of cardiac muscle. In this study, we analyze how the characteristics of spontaneous and triggered calcium sparks are related to cardiac ryanodine receptor (RYR) gating. We show that the frequency of spontaneous sparks and the probability distribution of calcium release flux quanta of triggered sparks correspond quantitatively to predictions of an allosteric homotetrameric model of RYR gating. This model includes competitive binding of Ca(2+) and Mg(2+) ions to the RYR activation sites and allosteric interaction between divalent ion binding and channel opening. It turns out that at rest, RYRs are almost fully occupied by Mg(2+). Therefore, spontaneous sparks are most frequently evoked by random openings of the highly populated but rarely opening Mg(4)RYR and CaMg(3)RYR forms, whereas triggered sparks are most frequently evoked by random openings of the less populated but much more readily opening Ca(2)Mg(2)RYR and Ca(3)MgRYR forms. In both the spontaneous and the triggered sparks, only a small fraction of RYRs in the calcium release unit manages to open during the spark because of the limited rate of Mg(2+) unbinding. This mechanism clarifies the unexpectedly low calcium release flux during elementary release events and unifies the theory of calcium signaling in resting and contracting cardiac myocytes.
- Published
- 2010
- Full Text
- View/download PDF
39. A cell architecture modeling system based on quantitative ultrastructural characteristics.
- Author
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Parulek J, Srámek M, Cerveanský M, Novotová M, and Zahradník I
- Subjects
- Animals, Humans, Organelles ultrastructure, Computer Simulation, Imaging, Three-Dimensional, Models, Biological, Muscle Cells ultrastructure
- Abstract
The architecture of living cells is difficult to describe and communicate; therefore, realistic computer models may help their understanding. 3D models should correspond both to qualitative and quantitative experimental data and therefore should include specific authoring tools such as appropriate visualization and stereological measures. For this purpose we have developed a problem solving environment for stereology-based modeling (PSE-SBM), which is an automated system for quantitative modeling of cell architecture. The PSE-SBM meets the requirement to produce models that correspond in stereological and morphologic terms to real cells and their organelles. Instead of using standard interactive graphing tools, our approach relies on functional modeling. We have built a system of implicit functions and set operations, organized in a hierarchical tree structure, which describes individual cell organelles and their 3D relations. Natural variability of size, shape, and position of organelles is achieved by random variation of the specific parameters within given limits. The resulting model is materialized by evaluation of these functions and is adjusted for a given set of specific parameters defined by the user. These principles are explained in detail, and modeling of segments of a muscle cell is used as an example to demonstrate the potential of the PSE-SBM for communication of architectural concepts and testing of structural hypotheses.
- Published
- 2009
- Full Text
- View/download PDF
40. Local calcium release activation by DHPR calcium channel openings in rat cardiac myocytes.
- Author
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Poláková E, Zahradníková A Jr, Pavelková J, Zahradník I, and Zahradníková A
- Subjects
- Animals, Cells, Cultured, Male, Rats, Rats, Wistar, Action Potentials physiology, Calcium metabolism, Calcium Channels, L-Type physiology, Calcium Signaling physiology, Ion Channel Gating physiology, Membrane Potentials physiology, Myocytes, Cardiac physiology
- Abstract
The principal role of calcium current in the triggering of calcium release in cardiac myocytes is well recognized. The mechanism of how calcium current (I(Ca)) controls the intensity of calcium release is not clear because of the stochastic nature of voltage-dependent gating of calcium channels (DHPRs) and of calcium-dependent gating of ryanodine receptors (RyRs). To disclose the relation between DHPR openings and the probability of calcium release, local calcium release activation by I(Ca) was investigated in rat ventricular myocytes using patch-clamp and confocal microscopy. Calcium spikes were activated by temporally synchronized DHPR calcium current triggers, generated by instantaneous 'tail' I(Ca) and modulated by prepulse duration, by tail potential, and by the DHPR agonist BayK 8644. The DHPR-RyR coupling fidelity was determined from the temporal distribution of calcium spike latencies using a model based on exponentially distributed DHPR open times. The analysis provided a DHPR mean open time of approximately 0.5 ms, RyR activation time constant of approximately 0.6 ms, and RyR activation kinetics of the 4th order. The coupling fidelity was low due to the inherent prevalence of very short DHPR openings but was increased when DHPR openings were prolonged by BayK 8644. The probability of calcium release activation was high, despite low coupling fidelity, due to the activation of many DHPRs at individual release sites. We conclude that the control of calcium release intensity by physiological stimuli can be achieved by modulating the number and duration of DHPR openings at low coupling fidelity, thus avoiding the danger of inadvertently triggering calcium release events.
- Published
- 2008
- Full Text
- View/download PDF
41. Inhibition of the cardiac L-type calcium channel current by antidepressant drugs.
- Author
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Zahradník I, Minarovic I, and Zahradníková A
- Subjects
- Animals, Dose-Response Relationship, Drug, Male, Membrane Potentials drug effects, Membrane Potentials physiology, Myocytes, Cardiac drug effects, Rats, Rats, Wistar, Antidepressive Agents pharmacology, Calcium Channel Blockers pharmacology, Calcium Channels, L-Type physiology, Myocytes, Cardiac physiology
- Abstract
Antidepressants inhibit many membrane receptors and ionic channels, including the L-type calcium channel. Here, we investigated the inhibition of calcium current (I(Ca)) by antidepressants in enzymatically isolated rat ventricular myocytes using whole-cell patch clamp. The molecular mechanism of inhibition was studied by comparing the voltage and state dependence of antidepressant inhibition of I(Ca) to the respective properties of calcium antagonists, and by studying the effect of (+/-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K8644) or diltiazem on the inhibitory potency of the antidepressants. All selected antidepressants inhibited calcium currents reversibly and concentration-dependently. At a stimulation frequency of 0.33 Hz, the antidepressants imipramine, clomipramine, desipramine, amitriptyline, maprotiline, citalopram, and dibenzepin blocked I(Ca), with IC(50) values of 8.3, 11.6, 11.7, 23.2, 31.0, 64.5, and 364 muM. The antidepressant drugs shifted steady-state inactivation curves of I(Ca) to negative voltages. The extent of the shift was similar to that induced by diltiazem or verapamil, but it was significantly smaller than that induced by felodipine. The use-dependent component of the antidepressant-induced block was similar to that of diltiazem, and it was significantly more and less, respectively, than those of felodipine and verapamil. In the presence of Bay K8644, antidepressants were more effective in inhibiting I(Ca). However, the inhibitory effect of antidepressants was also augmented by diltiazem, suggesting that these drugs do not compete with diltiazem for a single binding site. These data suggest that antidepressants exert their inhibitory action on cardiac L-type calcium channels by a specific interaction at a receptor site similar to, but distinct from, the benzothiazepine site.
- Published
- 2008
- Full Text
- View/download PDF
42. Evaluation of changes in secretory granules of atrial myocytes: a morphometric approach.
- Author
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Tylková L, Novotová M, Zahradník I, and Kiss A
- Subjects
- Animals, Microscopy, Electron, Transmission, Rats, Rats, Inbred Lew, Rats, Sprague-Dawley, Sensitivity and Specificity, Heart Atria ultrastructure, Myocytes, Cardiac ultrastructure, Secretory Vesicles ultrastructure
- Abstract
Objective: To evaluate the sensitivity and applicability of stereologic analysis for estimating changes in the secretory granule content of atrial myocytes., Study Design: The content of secretory granules in the right atrial myocytes of Sprague-Dawley (SD) and Lewis (LW) rats was assessed using a stereologic analysis of electron microscopic images under control conditions and in response to forced wheel running., Results: Volume density analysis revealed significant heterogeneity in the granule content of different strains of rats. The content of the dark secretory granules was significantly lower in control LW compared with control SD rats. The difference in pale granule content was opposite but less pronounced. Forced wheel running did not elicit statistically significant changes in the granule volume density. However, it changed significantly the number of dark granules in each rat strain, albeit in opposite directions, most likely due to changes in the number of small dark granules. No change was observed in the case of pale granules. This suggests higher sensitivity of dark granules to enhanced physical load., Conclusion: Both the volume and the number of secretory granules should be estimated in parallel to reveal responses of the atrial secretory system to different internal or external stimuli.
- Published
- 2008
43. Competitive and cooperative effects of Bay K8644 on the L-type calcium channel current inhibition by calcium channel antagonists.
- Author
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Zahradníková A, Minarovic I, and Zahradník I
- Subjects
- 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester metabolism, Algorithms, Allosteric Regulation, Animals, Binding Sites, Binding, Competitive, Calcium metabolism, Calcium Channel Agonists metabolism, Calcium Channel Agonists pharmacology, Calcium Channel Blockers metabolism, Cells, Cultured, Diltiazem pharmacology, Dose-Response Relationship, Drug, Drug Interactions, Felodipine pharmacology, Ion Channel Gating drug effects, Ion Channel Gating physiology, Male, Membrane Potentials drug effects, Models, Biological, Myocytes, Cardiac drug effects, Myocytes, Cardiac metabolism, Myocytes, Cardiac physiology, Patch-Clamp Techniques, Protein Binding, Rats, Rats, Wistar, Verapamil pharmacology, 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester pharmacology, Calcium Channel Blockers pharmacology, Calcium Channels, L-Type physiology
- Abstract
Phenylalkylamines, benzothiazepines, and dihydropyridines bind noncompetitively to the L-type calcium channel. The molecular mechanisms of this interaction were investigated in enzymatically isolated rat ventricular myocytes using the whole-cell patch-clamp technique. When applied alone, felodipine, verapamil, and diltiazem inhibited the L-type calcium current with values of inhibitory constant (K(B)) of 11, 246, and 512 nM, respectively, whereas 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K8644) activated I(Ca) with activation constant (K(A)) of 33 nM. Maximal activation of I(Ca) by 300 nM Bay K8644 strongly reduced the inhibitory potency of felodipine (apparent K(B) of 165 nM), significantly reduced the inhibitory potency of verapamil (apparent K(B) of 737 nM), but significantly increased the inhibitory potency of diltiazem (apparent K(B) of 310 nM). In terms of a new pseudoequilibrium two-drug binding model, the interaction between the dihydropyridine agonist Bay K8644 and the antagonist felodipine was found purely competitive. The interaction between Bay K8644 and verapamil or diltiazem was found noncompetitive, and it could be described only by inclusion of a negative interaction factor nu = -0.60 for verapamil and a positive interaction factor nu = +0.24 for diltiazem. These results suggest that at physiological membrane potentials, the L-type calcium channel cannot be simultaneously occupied by a dihydropyridine agonist and antagonist, whereas it can simultaneously bind a dihydropyridine agonist and a nondihydropyridine antagonist. Generally, the effects of the drugs on the L-type calcium channel support a concept of a channel domain responsible for binding of calcium channel antagonists and agonists changing dynamically with the membrane voltage and occupancy of individual binding sites.
- Published
- 2007
- Full Text
- View/download PDF
44. BLM Analyzer: a software tool for experiments on planar lipid bilayers.
- Author
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Novák P, Gaburjáková M, and Zahradník I
- Subjects
- Algorithms, Animals, Computational Biology methods, Electric Capacitance, Electrochemistry methods, Electrophysiology, Humans, Models, Statistical, Phospholipids chemistry, Reproducibility of Results, Software, Time Factors, Biosensing Techniques, Lipid Bilayers chemistry
- Abstract
Planar lipid bilayers represent a versatile platform for studying the functions of various membrane proteins as well as the development of biosensors. Despite the continuing technological progress in the fabrication of low-noise bilayer setups with mechanically and electrically stable planar bilayers, there is still a lack of software utilities for assistance during bilayer formation. We present here a multipurpose software tool, the bilayer lipid membrane (BLM) Analyzer which performs high-resolution measurements of bilayer capacitance and resistance using saw-tooth voltage stimulation. Based on the measured values of capacitance and resistance, the BLM Analyzer detects formation, stabilization, and breakage of lipid bilayer, automatically selects appropriate stimulus protocol, compensates for voltage offsets, and issues sound and voice alerts informing about the state of the measurement cycle. The principle of the BLM Analyzer is based on the integration of current responses within four equivalent time segments. It provides capacitance estimates with standard deviation of several femtofarads at temporal resolution of several tens of milliseconds. The functions of the BLM Analyzer were tested experimentally by monitoring formation and thinning of planar lipid bilayer.
- Published
- 2007
- Full Text
- View/download PDF
45. Kinetics of calcium spikes in rat cardiac myocytes.
- Author
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Zahradníková A Jr, Poláková E, Zahradník I, and Zahradníková A
- Subjects
- Animals, Calcium metabolism, Fluorescent Dyes, Male, Models, Theoretical, Rats, Rats, Wistar, Ryanodine Receptor Calcium Release Channel physiology, Time Factors, Calcium Signaling physiology, Membrane Potentials physiology, Myocytes, Cardiac physiology
- Abstract
The local calcium release flux signals (calcium spikes) evoked by membrane depolarization were recorded at high temporal resolution (2000 lines s(-1)) in isolated ventricular myocytes of male rats, using combination of scanning confocal microscopy and the patch-clamp technique. The kinetic properties of calcium spikes were investigated. The time course of calcium spike activation could be described reliably by a model with higher-order (n = 3) kinetics, but not by a first-order exponential process. A model of calcium spike with calcium release termination coupled to its activation was preferential to a model with the release termination independent of its activation. Three fluorescent calcium dyes (OG-5N, fluo-3, and fluo-4) were compared for calcium spike measurements. Experimental measurements as well as simulations showed that the occurrence and latency of calcium spikes could be measured faithfully with all indicators, while the kinetics of calcium spikes was reliably traced only with OG-5N. Calcium spikes evoked by a step depolarization from -50 to 0 mV commenced with a mean latency of 4.1 +/- 0.2 ms and peaked 6.7 +/- 0.2 ms later. Their full amplitudes were normally distributed. The activation time constant of calcium spikes was 3.1 +/- 0.1 ms, and the time constant of termination was 5.5 +/- 0.2 ms. A negative correlation was observed between the observed amplitude of calcium spikes and their time constant of activation, but there was no correlation between their observed amplitude and time constant of termination, in agreement with the concept of steep calcium-dependent activation and fateful inactivation of calcium release flux.
- Published
- 2007
- Full Text
- View/download PDF
46. Spatial and temporal Ca2+, Mg2+, and ATP2- dynamics in cardiac dyads during calcium release.
- Author
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Valent I, Zahradníková A, Pavelková J, and Zahradník I
- Subjects
- Animals, Computer Simulation, Cytosol metabolism, Diffusion, Humans, Kinetics, Research Design, Ryanodine Receptor Calcium Release Channel metabolism, Adenosine Triphosphate metabolism, Calcium Signaling, Magnesium metabolism, Models, Cardiovascular, Myocytes, Cardiac metabolism, Sarcoplasmic Reticulum metabolism
- Abstract
We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP(2-) with Ca(2+) and Mg(2+) ions on spatiotemporal concentration profiles of Ca(2+), Mg(2+), and ATP(2-) in the dyadic cleft during Ca(2+) release. The model revealed that Ca(2+) concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca(2+)] in the dyadic space reached values similar to estimates of luminal [Ca(2+)] in approximately 1 ms, suggesting that during calcium release the Ca(2+) gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca(2+) bound to ATP(2-), and thus substantially decreased ATP(2-) concentration in the dyadic space. The released Ca(2+) could also replace Mg(2+) in its complex with ATP(2-) during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca(2+), Mg(2+), and ATP(2-) might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.
- Published
- 2007
- Full Text
- View/download PDF
47. Ultrastructural remodeling of fast skeletal muscle fibers induced by invalidation of creatine kinase.
- Author
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Novotová M, Pavlovicová M, Veksler VI, Ventura-Clapier R, and Zahradník I
- Subjects
- Adaptation, Physiological, Animals, Creatine Kinase genetics, Isoenzymes genetics, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mitochondria metabolism, Mitochondria ultrastructure, Muscle, Skeletal cytology, Muscle, Skeletal metabolism, Creatine Kinase metabolism, Energy Metabolism, Isoenzymes metabolism, Muscle Fibers, Fast-Twitch enzymology, Muscle Fibers, Fast-Twitch ultrastructure
- Abstract
Understanding muscle adaptation to various stimuli is difficult because of the complex nature of stimuli and responses. In particular, responses to perturbations in energy metabolism require careful examination, because they may involve both structural and functional elements. To estimate the structural component of the myocyte adaptation to energetic deficiency, we used transgenic mice with blocked expression of mitochondrial and cytosolic creatine kinases (CK). The ultrastructure was analyzed using the stereological method of vertical sections applied to electron microscopic images of ultrathin longitudinal sections of fast muscle fibers of gastrocnemius, known to adapt to CK deficiency by increasing oxidative metabolism. The lack of CK induced a profound structural adaptation response that included changes in the volume and surface densities of major organelles. In addition, using a new stereological parameter, the environment of an organelle, substantial changes in the mitochondrial neighborhood were identified pointing to their relocation closer to the major sites of energy consumption, supposedly to compensate for invalidated energy transfer. Using quantitative arguments, we have shown for the first time that spatial relations among organelles of muscle cells undergo adaptation in response to nonstructural stimuli like metabolic deficiency.
- Published
- 2006
- Full Text
- View/download PDF
48. Q-method for high-resolution, whole-cell patch-clamp impedance measurements using square wave stimulation.
- Author
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Novák P and Zahradník I
- Subjects
- Cell Membrane physiology, Electric Impedance, Electrophysiology methods, Exocytosis physiology, Fertilization physiology, Ion Channel Gating physiology, Models, Biological, Patch-Clamp Techniques methods
- Abstract
High-resolution measurements of cell impedance provide invaluable information on various cellular processes such as exocytosis, ion channel gating, or fertilization. The best recent techniques, although achieving impedance resolution at theoretical limits, have limited applicability due to their inherent constrains and high complexity. We report here a simple method of high-resolution impedance measurement, dubbed as the Q-method, based on measurement of a charge by integrating the cell current during square wave stimulation and on its decomposition into specific components related to segments of the voltage stimulus. Simple relations were derived allowing very fast and direct estimation of cell impedance parameters. The major advantages of the Q-method are its inherently low sensitivity to low-pass filtering, rejection of periodic interference signals, automatic on-the-fly adjustment of the stimulation frequency for the highest capacitance resolution, and simultaneous high-resolution low-crosstalk monitoring of membrane resistance, series resistance and parasitic capacitance in addition to membrane capacitance. Implementation of the Q-method is straightforward with any patch-clamp setup and any cell type. Theoretical grounds of the Q-method, including its resolution and the noise of individual parameters, are developed and experimentally verified.
- Published
- 2006
- Full Text
- View/download PDF
49. Calcium activation of ryanodine receptor channels--reconciling RyR gating models with tetrameric channel structure.
- Author
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Zahradník I, Györke S, and Zahradníková A
- Subjects
- Allosteric Regulation physiology, Binding Sites drug effects, Binding Sites physiology, Calcium physiology, Dose-Response Relationship, Drug, Ion Channel Gating drug effects, Models, Biological, Mutant Proteins drug effects, Mutant Proteins physiology, Ryanodine Receptor Calcium Release Channel drug effects, Ryanodine Receptor Calcium Release Channel physiology, Calcium pharmacology, Ion Channel Gating physiology, Models, Theoretical, Ryanodine Receptor Calcium Release Channel chemistry
- Abstract
Despite its importance and abundance of experimental data, the molecular mechanism of RyR2 activation by calcium is poorly understood. Recent experimental studies involving coexpression of wild-type (WT) RyR2 together with a RyR2 mutant deficient in calcium-dependent activation (Li, P., and S.R. Chen. 2001. J. Gen. Physiol. 118:33-44) revealed large variations of calcium sensitivity of the RyR tetramers with their monomer composition. Together with previous results on kinetics of Ca activation (Zahradníková, A., I. Zahradník, I. Györke, and S. Györke. 1999. J. Gen. Physiol. 114:787-798), these data represent benchmarks for construction and testing of RyR models that would reproduce RyR behavior and be structurally realistic as well. Here we present a theoretical study of the effects of RyR monomer substitution by a calcium-insensitive mutant on the calcium dependence of RyR activation. Three published models of tetrameric RyR channels were used either directly or after adaptation to provide allosteric regulation. Additionally, two alternative RyR models with Ca binding sites created jointly by the monomers were developed. The models were modified for description of channels composed of WT and mutant monomers. The parameters of the models were optimized to provide the best approximation of published experimental data. For reproducing the observed calcium dependence of RyR tetramers containing mutant monomers (a) single, independent Ca binding sites on each monomer were preferable to shared binding sites; (b) allosteric models were preferable to linear models; (c) in the WT channel, probability of opening to states containing a Ca2+-free monomer had to be extremely low; and (d) models with fully Ca-bound closed states, additional to those of an Monod-Wyman-Changeaux model, were preferable to models without such states. These results provide support for the concept that RyR activation is possible (albeit vanishingly small in WT channels) in the absence of Ca2+ binding. They also suggest further avenues toward understanding RyR gating.
- Published
- 2005
- Full Text
- View/download PDF
50. Computer-aided formation of the whole-cell patch-clamp recording configuration.
- Author
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Novák P and Zahradník I
- Subjects
- Algorithms, Animals, Automation, Computers, Male, Models, Statistical, Rats, Rats, Wistar, Software, Electrophysiology methods, Heart Ventricles cytology, Muscle Cells cytology, Patch-Clamp Techniques instrumentation, Patch-Clamp Techniques methods, Signal Processing, Computer-Assisted
- Abstract
The conventional patch-clamp technique requires well-trained experimenter. Few commercial automated patch-clamp systems, designed for drug development, are better suited for large-scale research then for standard electrophysiological experiments. Here we describe a state machine for automated recognition of recording states of the patch-clamp experiment. The principle of the state machine is based on evaluation of the charge carried by membrane current during specific time segments in responses to square wave voltage stimulation. The state machine may serve for generating various sound alerts, signals for automated control of other devices, assistance in micromanipulation, internal pipette pressure control, and holding potential adjustments. Algorithm of the state machine, designed to cover wide variety of cell types, was successfully tested on rat ventricular myocytes.
- Published
- 2005
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