SOLUTION OF THE CROSS-TALK PROBLEM IN CELL IMPEDANCE ANALYSIS OF CARDIAC MYOCYTES

Membrane capacitance is a fundamental electrical characteristic of the surface membrane of living cells. The membrane capacitance is quantitatively related to the surface area, thickness and dielectric properties of the cell membrane and thus provides valuable information about the state of the cell. A generally accepted method for measuring membrane capacitance is based on stimulation of the cell with rectangular voltage pulses and approximation of the recorded membrane current by a mono-exponential decay function. We found that in cardiac muscle cells this method provides high variability of the measured capacitance and large cross-correlation among parameters of the measured circuit. In this study we focused on the elimination of cross-correlation error between the membrane capacitance, the membrane resistance, and the access resistance of the recording set-up. We showed how the use of the standard approximation model affects the level of crosstalk between estimates of these parameters. We proposed a modified model and tested its applicability on simulated and experimental data. The results revealed that the crosstalk error can be reduced by three orders of magnitude, well below the natural variability of membrane capacitance arising from biological reasons in cardiac myocytes

Spatial and temporal Ca2+, Mg2+, and ATP2− dynamics in cardiac dyads during calcium release

AbstractWe 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 ATP2− with Ca2+ and Mg2+ ions on spatiotemporal concentration profiles of Ca2+, Mg2+, and ATP2− in the dyadic cleft during Ca2+ release. The model revealed that Ca2+ concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca2+] in the dyadic space reached values similar to estimates of luminal [Ca2+] in ∼1 ms, suggesting that during calcium release the Ca2+ gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca2+ bound to ATP2−, and thus substantially decreased ATP2− concentration in the dyadic space. The released Ca2+ could also replace Mg2+ in its complex with ATP2− during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca2+, Mg2+, and ATP2− 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

Calcium Activation of Ryanodine Receptor Channels—Reconciling RyR Gating Models with Tetrameric Channel Structure

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

Calcium Signaling and Contractility in Cardiac Myocyte of Wolframin Deficient Rats

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

Assessment of methods for measuring dust emissions in livestock production

The occurrence of dust particles suspended in the air is a problem not only in increased concentration in the vicinity of human dwellings, but also for livestock. The theoretical part of this thesis is focused on the dust with a focus on current legislation and the impact on human and animal organism. Based on the robust simulation were performed dust measurements at two different commodities livestock. With impinger DUSTTRAK was detected current dust and compared according to the specified phase beem day

Reconstruction of membrane current by deconvolution and its application to membrane capacitance measurements in cardiac myocytes.

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

Geometrical Modelling of muscle cells based on functional representation of polygons

A geometrical modelling tool allowing construction of models of living cells and their organelles would facilitate understanding of biological processes at the cellular level. Here we describe a technique of skeletal muscle cells modelling, and a construction of the model based on the theory of implicit surfaces and their binary operations. The geometry of the cell is defined by means of parallel modelling planes perpendicular to its longitudinal axis. In each plane, the number, shape and topology of cell organelles is defined by means of functionally represented polygons. The model is obtained by interpolation between the planes. This approach allows to generate approximations of surfaces and to estimate the volume and surface densities of organelles in the model

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