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White-nose syndrome (WNS) is a fungal disease caused by

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans that is devastating to Nearctic bat populations but tolerated by Palearctic bats. Temperature is a factor known to be important for fungal growth and bat choice of hibernation. Here we investigated the effect of temperature on the pathogenic fungal growth in the wild across the Palearctic. We modelled body surface temperature of bats with respect to fungal infection intensity and disease severity and were able to relate this to the mean annual surface temperature at the site. Bats that hibernated at lower temperatures had less fungal growth and fewer skin lesions on their wings. Contrary to expectation derived from laboratory P. destructans culture experiments, natural infection intensity peaked between 5 and 6°C and decreased at warmer hibernating temperature. We made predictive maps based on bat species distributions, temperature and infection intensity and disease severity data to determine not only where P. destructans will be found but also where the infection will be invasive to bats across the Palearctic. Together these data highlight the mechanistic model of the interplay between environmental and biological factors, which determine progression in a wildlife disease.

Recent data on structure of dyads in cardiac myocytes indicate variable clustering of RyR calcium release channels. The question arises as to how geometric factors of RyR arrangement translate to their role in formation of calcium release events (CRE). Since this question is not experimentally testable in situ, we performed in silico experiments on a large set of calcium release site (CRS) models. The models covered the range of RyR spatial distributions observed in dyads, and included gating of RyRs with open probability dependent on Ca2+ and Mg2+ concentration. The RyR single-channel calcium current, varied in the range of previously reported values, was set constant in the course of CRE simulations. Other known features of dyads were omitted in the model formulation for clarity. CRE simulations initiated by a single random opening of one of the RyRs in a CRS produced spark-like responses with characteristics that varied with RyR vicinity, a newly defined parameter quantifying spatial distribution of RyRs in the CRSs, and with the RyR single-channel calcium current. The CRE characteristics followed the law of mass action with respect to a CRS state variable, defined as a weighed product of RyR vicinity and RyR single-channel calcium current. The results explained the structure-function relations among determinants of cardiac dyads on synergy principles and thus allowed to evolve the concept of CRS as a dynamic unit of cardiac dyad.

Iaparov et al. use in silico modeling to test the effect of changes in the geometric arrangement of RYR channels on calcium release in cardiac myocytes. Their simulations predict a coupling between RYR distribution at the calcium release site and dyad function., The dyads of cardiac myocytes contain ryanodine receptors (RYRs) that generate calcium sparks upon activation. To test how geometric factors of RYR distribution contribute to the formation of calcium sparks, which cannot be addressed experimentally, we performed in silico simulations on a large set of models of calcium release sites (CRSs). Our models covered the observed range of RYR number, density, and spatial arrangement. The calcium release function of CRSs was modeled by RYR openings, with an open probability dependent on concentrations of free Ca2+ and Mg2+ ions, in a rapidly buffered system, with a constant open RYR calcium current. We found that simulations of spontaneous sparks by repeatedly opening one of the RYRs in a CRS produced three different types of calcium release events (CREs) in any of the models. Transformation of simulated CREs into fluorescence signals yielded calcium sparks with characteristics close to the observed ones. CRE occurrence varied broadly with the spatial distribution of RYRs in the CRS but did not consistently correlate with RYR number, surface density, or calcium current. However, it correlated with RYR coupling strength, defined as the weighted product of RYR vicinity and calcium current, so that CRE characteristics of all models followed the same state-response function. This finding revealed the synergy between structure and function of CRSs in shaping dyad function. Lastly, rearrangements of RYRs simulating hypothetical experiments on splitting and compaction of a dyad revealed an increased propensity to generate spontaneous sparks and an overall increase in calcium release in smaller and more compact dyads, thus underlying the importance and physiological role of RYR arrangement in cardiac myocytes.

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.

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.

In underground hibernacula temperate northern hemisphere bats are exposed to Pseudogymnoascus destructans, the fungal agent of white-nose syndrome. While pathological and epidemiological data suggest that Palearctic bats tolerate this infection, we lack knowledge about bat health under pathogen pressure. Here we report blood profiles, along with body mass index (BMI), infection intensity and hibernation temperature, in greater mouse-eared bats (Myotis myotis). We sampled three European hibernacula that differ in geomorphology and microclimatic conditions. Skin lesion counts differed between contralateral wings of a bat, suggesting variable exposure to the fungus. Analysis of blood parameters suggests a threshold of ca. 300 skin lesions on both wings, combined with poor hibernation conditions, may distinguish healthy bats from those with homeostatic disruption. Physiological effects manifested as mild metabolic acidosis, decreased glucose and peripheral blood eosinophilia which were strongly locality-dependent. Hibernating bats displaying blood homeostasis disruption had 2 °C lower body surface temperatures. A shallow BMI loss slope with increasing pathogen load suggested a high degree of infection tolerance. European greater mouse-eared bats generally survive P. destructans invasion, despite some health deterioration at higher infection intensities (dependant on hibernation conditions). Conservation measures should minimise additional stressors to conserve constrained body reserves of bats during hibernation.

Summary Specimens archived in wet collections represent valuable material for scientific research. Here, we show that bat fly (Diptera, Nycteribiidae) samples contain DNA of Pseudogymnoascus destructans, a fungus pathogenic to bats. Using dual-probe quantitative PCR, we detected P. destructans DNA on bat flies collected in the Samara, Sverdlovsk and Irkutsk regions of Russia between 2005 and 2017. Fungal load was significantly lower on bat flies from wet collections than on freshly collected mites in the Czech Republic. The bat pathogen was present in the Samara region (European part of Russia) in 2005, that is, a year before recognition of white-nose syndrome in North America. As Samara and Irkutsk regions were identified as new positive locations of P. destructans, our data expand the known geographic distribution of P. destructans. We conclude that ethanol-stored ectoparasites can be used to identify the presence of pathogens in historic bat populations and understudied geographical regions.

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.

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, PO, 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. (C) 2020 Elsevier Ltd. All rights reserved.

Calcium release sites (CRSs) play a key role in excitation–contraction coupling of cardiac myocytes. Recent studies based on electron tomography and super-resolution imaging revealed that CRSs are not completely filled with ryanodine receptors (RyRs) and that the spatial arrangement of RyRs is neither uniform nor static. In this work, we studied the effect of spatial arrangement of RyRs on RyR activation using simulations based on Monte Carlo (MC) and mean-field (MF) approaches. Both approaches showed that activation of RyRs is sensitive to the arrangement of RyRs in the CRS. However, the MF simulations did not reproduce results of MC simulations for non-compact CRSs, suggesting that the approximations used in the MF approach are not suitable for simulation studies of RyRs arrangements observed experimentally. MC simulations revealed the importance of realistic spatial arrangement of RyRs for adequate modelling of calcium release in cardiac myocytes.

Background Hibernation allows animals to survive periods of resource scarcity by reducing their energy expenditure through decreased metabolism. However, hibernators become susceptible to psychrophilic pathogens if they cannot mount an efficient immune response to infection. While Nearctic bats infected with white-nose syndrome (WNS) suffer high mortality, related Palearctic taxa are better able to survive the disease than their Nearctic counterparts. We hypothesised that WNS exerted historical selective pressure in Palearctic bats, resulting in genomic changes that promote infection tolerance. Results We investigated partial sequences of 23 genes related to water metabolism and skin structure function in nine Palearctic and Nearctic hibernating bat species and one non-hibernating species for phylogenetic signals of natural selection. Using maximum likelihood analysis, we found that eight genes were under positive selection and we successfully identified amino acid sites under selection in five encoded proteins. Branch site models revealed positive selection in three genes. Hibernating bats exhibit signals for positive selection in genes ensuring tissue regeneration, wound healing and modulation of the immune response. Conclusion Our results highlight the importance of skin barrier integrity and healing capacity in hibernating bats. The protective role of skin integrity against both pathophysiology and WNS progression, in synergy with down-regulation of the immune reaction in response to the Pseudogymnoascus destructans infection, improves host survival. Our data also suggest that hibernating bat species have evolved into tolerant hosts by reducing the negative impact of skin infection through a set of adaptations, including those at the genomic level.

Calcium waves can form and propagate at low frequencies of spontaneous calcium sparks if the calcium dependence of spark frequency is sufficiently steep, or the number of open RyRs is sufficiently large., The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05–0.1-µM) calcium concentrations but only slightly at high (0.2–0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases.

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.

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix., Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardio­myocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals Cα atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

The principal role of calcium current in the triggering of calcium release in cardiac myocytes is well recognized. The mechanism of how calcium current (ICa) 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 ICa 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’ICa 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 ∼0.5 ms, RyR activation time constant of ∼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.

The effect of a domain peptide DPCPVTc from the central region of the RYR2 on ryanodine receptors from rat heart has been examined in planar lipid bilayers. At a zero holding potential and at 8 mmol L−1 luminal Ca2+ concentration, DPCPVTc induced concentrationdependent activation of the ryanodine receptor that led up to 20-fold increase of PO at saturating DPCPVTc concentrations. DPCPVTc prolonged RyR2 openings and increased RyR2 opening frequency. At all peptide concentrations the channels displayed large variability in open probability, open time and frequency of openings. With increasing peptide concentration, the fraction of high open probability records increased together with their open time. The closed times of neither low- nor high-open probability records depended on peptide concentration. The concentration dependence of all gating parameters had EC50 of 20 μmol L−1 and a Hill slope of 2. Comparison of the effects of DPCPVTc with the effects of ATP and cytosolic Ca2+ suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DPCPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.

A striking feature of white-nose syndrome, a fungal infection of hibernating bats, is the difference in infection outcome between North America and Europe. Here we show high WNS prevalence both in Europe and on the West Siberian Plain in Asia. Palearctic bat communities tolerate similar fungal loads of Pseudogymnoascus destructans infection as their Nearctic counterparts and histopathology indicates equal focal skin tissue invasiveness pathognomonic for WNS lesions. Fungal load positively correlates with disease intensity and it reaches highest values at intermediate latitudes. Prevalence and fungal load dynamics in Palearctic bats remained persistent and high between 2012 and 2014. Dominant haplotypes of five genes are widespread in North America, Europe and Asia, expanding the source region of white-nose syndrome to non-European hibernacula. Our data provides evidence for both endemicity and tolerance to this persistent virulent fungus in the Palearctic, suggesting that host-pathogen interaction equilibrium has been established.

Proper function of cardiac myocytes requires a reliable mechanism for regulation of calcium influx during the contraction-relaxation cycle. The most effective is negative feedback based on the calcium-dependent inactivation of calcium current. Calcium release activated by L-type Ca-current causes rapid and extensive inactivation of the Ca-current. The aim of this study was to characterize the effect of local Ca-release on the extent of calcium release-dependent inactivation (RDI) using characteristics of calcium spikes.Calcium release was induced in isolated rat ventricular myocytes in the whole-cell patch-clamp configuration, using a two pulse protocol [1]. A short pre-pulse of varying length or voltage was followed by a standard test-pulse (0 mV, 80 ms). The extent of RDI was assessed as the fraction of peak Ca-current remaining in the test-pulse relative to peak current in the absence of the pre-pulse. Local calcium release fluxes (Ca-spikes) were measured by laser scanning fluorescence confocal microscopy using 0.1 mM fluo-3 and 1 mM EGTA in the patch-pipette solution. The latency and the amplitude were determined for individual Ca-spikes. Probability of Ca-release activation (PA) was calculated as the fraction of activated calcium release sites. Synchrony of Ca-release was calculated as the inverse of standard deviation of the latency.The extent of RDI showed linear correlation with PA (R = 0.95) and amplitude (R = 0.89), while latency and synhrony did not affect the extent of RDI. The extent of RDI showed sigmoidal dependence on the product of amplitude and PA that reflects the amount of released calcium. We conclude that RDI of L-type calcium currents results from the local character of calcium release and local calcium-dependent inactivation of L-type calcium channels.Support: VEGA 2/0095/15 and VEGA 2/0147/14[1] Zahradnikova et al., 2004. Am J Physiol Cell Physiol (286): 330-341

This study was supported through a grant from the Czech Science Foundation (Grant No. P506-12-1064). We are grateful to Tomas Bartonicka, Hana Berkova and Masha Orlova for invaluable field assistance, to Matej Dolinay, Jiri C. Moravec, Patricia Pecnerova and Aneta Reichova for laboratory assistance and to Gregory G. Turner for biopsy samples from Nearctic bats. A striking feature of white-nose syndrome, a fungal infection of hibernating bats, is the difference in infection outcome between North America and Europe. Here we show high WNS prevalence both in Europe and on the West Siberian Plain in Asia. Palearctic bat communities tolerate similar fungal loads of Pseudogymnoascus destructans infection as their Nearctic counterparts and histopathology indicates equal focal skin tissue invasiveness pathognomonic for WNS lesions. Fungal load positively correlates with disease intensity and it reaches highest values at intermediate latitudes. Prevalence and fungal load dynamics in Palearctic bats remained persistent and high between 2012 and 2014. Dominant haplotypes of five genes are widespread in North America, Europe and Asia, expanding the source region of white-nose syndrome to non-European hibernacula. Our data provides evidence for both endemicity and tolerance to this persistent virulent fungus in the Palearctic, suggesting that host-pathogen interaction equilibrium has been established.

The synergic effect of luminal Ca2+, cytosolic Ca2+, and cytosolic adenosine triphosphate (ATP) on activation of cardiac ryanodine receptor (RYR2) channels was examined in planar lipid bilayers. The dose–response of RYR2 gating activity to ATP was characterized at a diastolic cytosolic Ca2+ concentration of 100 nM over a range of luminal Ca2+ concentrations and, vice versa, at a diastolic luminal Ca2+ concentration of 1 mM over a range of cytosolic Ca2+ concentrations. Low level of luminal Ca2+ (1 mM) significantly increased the affinity of the RYR2 channel for ATP but without substantial activation of the channel. Higher levels of luminal Ca2+ (8–53 mM) markedly amplified the effects of ATP on the RYR2 activity by selectively increasing the maximal RYR2 activation by ATP, without affecting the affinity of the channel to ATP. Near-diastolic cytosolic Ca2+ levels (

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.

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.

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.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic disease characterized by stress-induced syncope and/or sudden death in young individuals with structurally normal heart. More than 150 mutations located in the cardiac Ca2+ release channel (type-2 ryanodine receptor, RyR2) gene are related to CPVT. Besides ventricular tachycardia (VT) under stress, sinoatrial node (SAN) dysfunction is frequently observed in CPVT patients. However, the cellular mechanisms remain underexplored. We created a KI mice model bearing a mutation in the N-terminal portion of the RyR2 found in a CPVT family, RyR2(R420Q). ECGs were recorded in KI and WT littermates in resting condition and after epinephrine/caffeine (2/120 mg/kg) challenge (i.p. injection). All KI mice (n=7) showed sustained bidirectional VT, validating the model. Moreover, resting heart rhythm was slower in KI mice during rest period (525.3±9.0 bpm n=4 and 498.5±4.9 bpm n=3, P

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.

Local calcium release via RYRs, observed in the form of calcium sparks ([Cheng et al., 1993][1]), is generally accepted to be a principal mediator of calcium homeostasis and excitation–contraction coupling in cardiac myocytes. However, investigation of local calcium release signals is extremely

In the heart, depolarization during the action potential activates voltage-dependent Ca2+ channels that mediate a small, localized Ca2+ influx (ICa). This small Ca2+ signal activates specialized Ca2+ release channels, the ryanodine receptors (RyRs), in the sarcoplasmic reticulum (SR). This process

The local control concept of excitation–contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca2+entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (2+elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca2+signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca2+generated by flash photolysis of DM-nitrophen. Application of such Ca2+spikes (amplitude ∼10–30 μM, duration ∼0.1–0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope ∼2.5) with spike amplitude. The time constants of RyR activation were 0.07–0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a powern∼ 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca2+-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca2+triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca2+ions in accordance with the tetrameric organization of the channel protein.

NO donors were found to reduce the rate of Ca2+ release from isolated skeletal muscle sarcoplasmic reticulum (SR) and the open probability of single ryanodine receptor Ca2+ release channels (RyRCs) in planar lipid bilayers, and these effects were prevented by the NO quencher hemoglobin and reversed by 2-mercaptoethanol. Ca2+ release assessed in skeletal muscle homogenates was also reduced by NO that was generated in situ from l-arginine by endogenous, nitro-l-arginine methylester-sensitive NO-synthase. The effect of NO on the RyRC might explain NO-induced depression of contractile force in striated muscles and, since both RyRC isoforms and NOS isoenzymes are ubiquitous, may represent a wide-spread feedback mechanism in Ca2+ signaling; i.e. Ca-dependent activation of NO production and NO-evoked reduction of Ca2+ release from intracellular Ca2+ stores.

Background— Phosphoinositide 3-kinase γ (PI3Kγ) signaling engaged by β-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3Kγ in catecholamine-induced arrhythmia is currently unknown. Methods and Results— Mice lacking PI3Kγ (PI3Kγ −/− ) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective β 2 -adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after β 2 -adrenergic receptor activation in PI3Kγ −/− cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3Kγ. Downstream from β-adrenergic receptors, PI3Kγ was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3Kγ-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Ca v 1.2) and phospholamban. In PI3Kγ −/− cardiomyocytes, Ca v 1.2 and phospholamban were hyperphosphorylated, leading to increased Ca 2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3Kγ −/− cardiomyocytes showed spontaneous Ca 2+ release events and developed arrhythmic calcium transients. Conclusions— PI3Kγ coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.

Ryanodine receptors (RyRs) are homotetrameric intracellular calcium release channels in the membranes of the endoplasmic (ER) and sarcoplasmic reticulum (SR) (George et al. 2005, Meissner 2002, 2004). Each subunit consists of ~5000 amino acid residues (George et al. 2005). There are three isoforms of the ryanodine receptor: the RyR1 isoform is expressed predominantly in skeletal muscle, the RyR2 isoform predominates in cardiac muscle, and the RyR3 isoform is expressed in a variety of tissues (Sorrentino 1995). In the mammalian heart, the RyR2 isoform is a principal component of the excitation-contraction (E-C) coupling process. Action potential depolarization of the cardiac cell results in injection of calcium ions into the cell via calcium channels (dihydropyridine receptors, DHPRs). This small calcium influx then drives the release of calcium from intracellular calcium stores by triggering the opening of RyR2 channels (Fabiato 1985). The released calcium causes contraction by binding to troponin C (Ebashi and Ogawa 1988). Consequently, precise regulation of RyR activity during heartbeat is essential to proper cardiac function. In several cardiac diseases, such as heart failure and the genetic diseases CVPT (catecholaminergic polymorphic ventricular tachycardia) and ARVD2 (arrhythmogenic right ventricular dysplasia), the function of RyR is compromised. In heart failure, the release of calcium in response to the action potential is decreased, while RyR remains more active during the diastole (Durham et al. 2007, Yano et al. 2006). In CPVT and ARVD2, RyRs contain mutations that lead to altered RyR activity which may result in premature calcium release in the absence of an action potential (Durham et al. 2007, Yano et al. 2006). In this work we present a bioinformatics analysis of the whole of human RyR2 (hRyR2) in context with the available functional information, in order to locate individual domains for further biochemical and structural studies. The reliability of the predictions in the Nterminal region (Bauerova-Hlinkova et al. 2010) was verified experimentally by expressing and characterizing the domains identified. We also describe the results of a CD

Calcium released from endoplasmic reticulum through special calcium release channels - inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) - serves as a main source of cytosolic calcium signaling in the majority of cell types in physiological state and also in pathological situations. In this work, we studied whether IP(3)Rs can be involved in uranyl acetate induced nephrotoxicity. Using human embryonic kidney cell line (HEK293) as an experimental model we have found that uranyl acetate (5 and 50microM) up-regulates both, mRNA and protein levels of the type 1 and type 2 IP(3) receptors in HEK293 cells. This increase was associated with elevated expression of proapoptotic factors Bax and Caspase 3 and also by higher extent of apoptosis. Vice versa, induction of apoptosis resulted in increased mRNA levels of IP(3)R2 and also elevated levels of apoptotic markers. Therefore we propose that enhanced expression of the type 2 IP(3)Rs can at least partially contribute to increased levels of apoptosis due to uranyl acetate treatment.

Type 1 and 2 inositol 1,4,5-trisphosphate (IP3) receptors have been found in cardiac tissue, although they are localized in different types of cells. While the type 1 predominates in neuronal cells and cardiac ganglia, type 2 IP3 receptor is localized mainly in cardiomyocytes. In the heart, gene expression of the type 1 IP3 receptor is modulated by catecholamines, while type 2 is not affected. In this study we compared the effects of two stressors--cold exposure and hypoxia--on gene expression and protein levels of type 1 and 2 IP3 receptors. We found that both types of IP3 receptors were increased by these stressors, although the response of the type 2 IP3 receptor was delayed. We propose that regulation of these types of IP3 receptors in the heart might differ in control conditions and during stress.

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.

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.

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.

Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3′-hydroxyl group of the DNA molecule during phosphodiester bond formation.

In mammalian cardiac myocytes, calcium released into the dyadic space rapidly inactivates calcium current ( ICa). We used this Ca2+ release-dependent inactivation (RDI) of ICa as a local probe of sarcoplasmic reticulum Ca2+ release activation. In whole cell patch-clamped rat ventricular myocytes, Ca2+ entry induced by short prepulses from —50 mV to positive voltages caused suppression of peak ICa during a test pulse. The negative correlation between peak ICa suppression and ICa inactivation during the test pulse indicated that RDI evoked by the prepulse affected only calcium channels in those dyads in which calcium release was activated. Ca2+ ions injected during the prepulse and during the subsequent tail current suppressed peak ICa in the test pulse to a different extent. Quantitative analysis indicated that equal Ca2+ charge was 3.5 times less effective in inducing release when entering during the prepulse than when entering during the tail. Tail Ca2+ charge injected by the first voltage-dependent calcium channel (DHPR) openings was three times less effective than that injected by DHPR reopenings. These findings suggest that calcium release activation can be profoundly influenced by the recent history of L-type Ca2+ channel activity due to potentiation of ryanodine receptors (RyRs) by previous calcium influx. This conclusion was confirmed at the level of single RyRs in planar lipid bilayers: using flash photolysis of the calcium cage NP-EGTA to generate two sequential calcium stimuli, we showed that RyR activation in response to the second stimulus was four times higher than that in response to the first stimulus.

Mg2+, an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca2+release channels by competing with Ca2+at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca2+-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca2+stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca2+compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg2+at the background of 3 mM total ATP in the presence or absence of 1 mM luminal Ca2+. Mg2+dramatically slowed the rate of activation of RyRs in response to sustained (≥10-ms) elevations in Ca2+concentration. Paradoxically, Mg2+had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (2+stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca2+did not affect the peak open probability or the probability of responses to brief Ca2+signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg2+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 Ca2+channels and RyRs in heart cells.

Human cardiac ryanodine receptor (hRyR2) is a channel mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling. The N-terminal (1-655) and central (2100-2500) regions of hRyR2 are thought to be involved in regulating channel gating. Mutations linked to several heart diseases are clustered within these two, as well as in the channel pore-containing C-terminal regions. High resolution structures of key regions involved in the regulation of RyR2 activity could further the understanding of the gating mechanism of hRyR2 and of its malfunction in disease.

The effect of a domain peptide DPCPVTc from the central region of the RYR2 on ryanodine receptors isolated from rat heart was examined in planar lipid bilayers. At a zero holding potential and at 100 nM cytosolic and 8 mM luminal Ca2+ concentration, DPCPVTc induced concentration-dependent activation of the ryanodine receptor that led up to 20-fold increase of open probability at saturating DPCPVTc concentrations. The effect of the peptide appeared within 30 s after addition to the experimental chamber. At all DPCPVTc concentrations RyR2 channels displayed large variability in open probability, open time and opening frequency. DPCPVTc prolonged RyR2 openings up to 8× and increased RyR2 opening frequency by up to 100%. With increasing DPCPVTc concentration, the fraction of high open probability records increased up to 5×, and their open time increased up to 4×. The closed times did not depend on DPCPVTc concentration either in low- or high-open probability records. The DPCPVTc concentration dependence of all gating parameters had EC50 of 20 µM and a Hill slope of 2. Comparison of the effects of DPCPVTc with those of ATP [1] and cytosolic Ca2+ [2] suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DPCPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.Supported in part by the European Union Contract No. LSHM-CT-2005-018802/CONTICA and by grants APVV-LPP-0441-09, APVV-0628-10 and VEGA 2/0197/11.[1] Tencerova B, Zahradnikova A, Gaburjakova J, Gaburjakova M. J Gen Physiol 140: 93, 2012.[2] Gaburjakova J, Gaburjakova M. J Membr Biol 212: 17, 2006.