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

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

Automatic assessment of the cardiomyocyte development stages from confocal microscopy images using deep convolutional networks

Computer assisted image acquisition techniques, including confocal microscopy, require efficient tools for an automatic sorting of vast amount of generated image data. The complexity of the classification process, absence of adequate tools, and insufficient amount of reference data has made the automated processing of images challenging. Mastering of this issue would allow implementation of statistical analysis in research areas such as in research on formation of t-tubules in cardiac myocytes. We developed a system aimed at automatic assessment of cardiomyocyte development stages (SAACS). The system classifies confocal images of cardiomyocytes with fluorescent dye stained sarcolemma. We based SAACS on a densely connected convolutional network (DenseNet) topology. We created a set of labelled source images, proposed an appropriate data augmentation technique and designed a class probability graph. We showed that the DenseNet topology, in combination with the augmentation technique is suitable for the given task, and that high-resolution images are instrumental for image categorization. SAACS, in combination with the automatic high-throughput confocal imaging, will allow application of statistical analysis in the research of the tubular system development or remodelling and loss

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

Quantitative analysis of calcium spikes in noisy fluorescent background.

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

Determination of the accuracy and the precision of the estimated parameters of OG-5N (A, B) and Fluo-3 spikes (C, D).

<p><b>A, C</b> – correlation between the simulated and the fitted parameters (grey circles). Red line is the linear fit. Parameters of the spikes with the bottom 5% of SNR values (2.38±0.04 for OG-5N and 4.33±0.15 for Fluo-3) and of spikes with the top 5% of SNR values (10.11±0.12 for OG-5N and 26.68±0.29 for Fluo-3) are shown as black and blue circles, respectively. Pearson correlation coefficients between the parameters of simulated OG-5N calcium spikes and parameters of their fits were 0.986, 0.893, 0.869 and 0.942 for the A, t₀, TTP and FDHM values, respectively. Pearson correlation coefficients between the parameters of simulated Fluo-3 calcium spikes and parameters of their fits were 0.999, 0.939, 0.962 and 0.993 for the A, t₀, TTP and FDHM values, respectively. <b>B, D</b> – mean ± s.d. of the fitted parameter values grouped by the SNR values of the simulated spikes. Black lines are the mean parameter values of the simulated spikes in the absence of noise. Red lines show mean+s.d. and mean - s.d. of parameter values of noise-free simulated spikes.</p