Localized Calcium Release Events Research Articles

Functionally linked potassium channel activity in cerebral endothelial and smooth muscle cells is compromised in Alzheimer’s disease

The brain microcirculation is increasingly viewed as a potential target for disease-modifying drugs in the treatment of Alzheimer's disease patients, reflecting a growing appreciation of evidence that cerebral blood flow is compromised in such patients. However, the pathogenic mechanisms in brain resistance arteries underlying blood flow defects have not yet been elucidated. Here we probed the roles of principal vasodilatory pathways in cerebral arteries using the APP23 mouse model of Alzheimer's disease, in which amyloid precursor protein is increased approximately sevenfold, leading to neuritic plaques and cerebrovascular accumulation of amyloid-β similar to those in patients with Alzheimer's disease. Pial arteries from APP23 mice (18 mo old) exhibited enhanced pressure-induced (myogenic) constriction because of a profound reduction in ryanodine receptor-mediated, local calcium-release events ("Ca2+ sparks") in arterial smooth muscle cells and a consequent decrease in the activity of large-conductance Ca2+-activated K+ (BK) channels. The ability of the endothelial cell inward rectifier K+ (Kir2.1) channel to cause dilation was also compromised. Acute application of amyloid-β 1-40 peptide to cerebral arteries from wild-type mice partially recapitulated the BK dysfunction seen in APP23 mice but had no effect on Kir2.1 function. If mirrored in human Alzheimer's disease, these tandem defects in K+ channel-mediated vasodilation could account for the clinical cerebrovascular presentation seen in patients: reduced blood flow and crippled functional hyperemia. These data direct future research toward approaches that reverse this dual vascular channel dysfunction, with the ultimate aim of restoring healthy cerebral blood flow and improving clinical outcomes.

Noise analysis of cytosolic calcium image data

Cellular Ca2+ signals are often constrained to cytosolic micro- or nano-domains where stochastic openings of Ca2+ channels cause large fluctuations in local Ca2+ concentration (Ca2+ ‘noise’). With the advent of TIRF microscopy to image the fluorescence of Ca2+-sensitive probes from attoliter volumes it has become possible to directly monitor these signals, which closely track the gating of plasmalemmal and ER Ca2+-permeable channels. Nevertheless, it is likely that many physiologically important Ca2+ signals are too small to resolve as discrete events in fluorescence recordings. By analogy with noise analysis of electrophysiological data, we explore here the use of statistical approaches to detect and analyze such Ca2+ noise in images obtained using Ca2+-sensitive indicator dyes. We describe two techniques - power spectrum analysis and spatio-temporal correlation - and demonstrate that both effectively identify discrete, spatially localized calcium release events (Ca2+ puffs). Moreover, we show they are able to detect localized noise fluctuations in a case where discrete events cannot directly be resolved.

Live Cell Palm Techniques for Super Resolution Imaging of Murine Cardiac Myocytes

Since their inception, super resolution methodologies have revolutionised the way in which biologists can visualise subcellular structures. Cardiac science has particularly benefited under these methodologies, as structures such as the dyads of cardiomyocytes have become resolvable, thus yielding new understanding of -their spatial organization. However, pairing of nanoscale dyadic structure and function has been hampered by the fact that techniques such as direct stochastic optical reconstruction microscopy (dSTORM) generally require fixed tissue. Furthermore, quantification of dSTORM images based on antibody labelling is complicated by variability in labelling efficacy, and displacement of the fluorophore from the protein of interest. We presently circumvented these limitations by creating a transgenic mouse expressing a photo activated red fluorescent protein tagged to ryanodine receptor 2 (PA-tag-RFP-RyR2). Imaging of live, isolated cardiomyocytes was attained with a commercial Zeiss Elyra dSTORM setup using the photo-activated light microscopy (PALM) technique. PA Tag RFP requires dual sample illumination with activation laser and imaging laser to produce constant photo switching. In our experiments we found that by adjusting the rate of activation and photobleaching from imaging we could optimize the blinking behaviour required for live cell PALM imaging. To reduce background, HILO and TIRF illumination were employed to reduce the thickness of the optical section. Our results show RyR localisation accuracy of 40nm in the X and Y plane for live cell images, in contrast to the higher resolution of 10-20nm in fixed samples with antibody labelling. RyR cluster arrangements in live cells were observed to be broadly in agreement with fixed samples. Ongoing work is aimed at linking RyR organization to local calcium release events (Ca2+ sparks), with focus on unravelling the functional consequences of the dynamic nature of RyR arrangement.

The Malignant Hyperthermia RYR1Y522S Mutation affects Calcium Homeostasis in Arterial Smooth Muscle Cells

Type 1 ryanodine receptor (RyR1) is a key protein involved in the regulation of the intracellular Ca2+ concentration in skeletal muscle cells, playing a crucial role in muscle contraction by releasing Ca2+ from the sarcoplasmic reticulum after plasma membrane depolarization. Dysregulation of calcium signals due to defects in RyR1 have been associated with a wide range of primary neuromuscular disorders, including Malignant Hyperthermia Susceptibility and a number of congenital myopathies including Core Myopathies, some forms of Centronuclear myopathy and Congenital Fiber Type Disproportion. Thus mutations in RYR1 (the gene encoding RyR1) may lead to alterations of Ca2+ homeostasis not only in skeletal muscle, but also in other tissues expressing this intracellular calcium release channel. In the present report we performed the biochemical characterization and immunofluorescence analysis of RyR1 in arterial smooth muscle cells. We found that smooth muscle cells isolated from the RYR1Y522S knock in mouse exhibited increased frequency of localized calcium release events (sparks) and smaller intracellular Ca2+ stores compared to WT littermates, although the resting calcium was not affected. In conclusion we show that a RYR1 mutation associated with Malignant Hyperthermia alters vascular smooth muscle cell function by affecting calcium homeostasis.

Effects of Triad Geometry and RyR Gating Scheme on Simulated Skeletal Muscle Sparks

Computer models of calcium sparks have been an important tool in their investigation, especially in understanding the way how the regenerative calcium signal is initiated and terminated.The aim of the present work was to use an existing 3D model with distributed sarcoplasmic reticulum (SR) for cardiac sparks and demonstrate the effects of altered calcium release unit (CRU) geometry and ryanodine receptor (RyR) gating on the generation and termination of calcium release events in skeletal muscle.First, only the geometry of CRU was modified to match the triad structure found in skeletal muscle cells. This was achieved by arranging RyRs in double rows in the terminal cisternae (TC) membrane on each side of the T-tubule and by reducing the diameter of the T-tubule. Such a model was unable to produce the regenerative phase of the calcium spark following the stochastic opening of a single RyR. Next, the gating of RyRs was modified by shifting its calcium sensitivity to the left to mimic the baseline activity of skeletal-type RyR. This model reproduced the rising phase and the peak of the spark, but was unable to model its complete termination as observed in events measured in Saponin-permeabilized skeletal muscle fibers. Refilling of the TC had to be severely constrained or more complex gating schemes had to be introduced in order to make the termination of simulated sparks similar to that seen in the experiments.These results indicate that both the exact geometry of CRU and the gating scheme of RyR contribute to the generation and termination of localized calcium release events to a great extent and, therefore, these details have to be taken into account when their characteristics and role are examined in skeletal muscle.

Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells.

The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This "calcium clock" takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic-mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating rate in response to β-adrenergic stimulation. The model indicates that the hierarchical clustering of surface RyRs in SANCs may be a crucial adaptive mechanism. Pathological desynchronization of the clocks may explain sinus node dysfunction in heart failure and RyR mutations.

Ryanodine receptors, calcium signaling, and regulation of vascular tone in the cerebral parenchymal microcirculation.

The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of CBF, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of PAs. There are, moreover, notable functional differences between pial arteries and PAs. For example, in pial VSMCs, local calcium release events ("calcium sparks") through ryanodine receptor (RyR) channels in SR membrane activate large conductance, calcium-sensitive potassium channels to modulate vascular diameter. In contrast, VSMCs in PAs express functional RyR and BK channels, but under physiological conditions, these channels do not oppose pressure-induced vasoconstriction. Here, we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of CBF.

Efficient Automatic Analysis of High-Speed Confocal Images Containing Localized Calcium Release Events

High-speed confocal microscopy is a relatively new method used to investigate localized calcium release events, most notably calcium sparks. Line scan images can reliably be recorded at speeds up to 60000 lines/s while for X-Y images 120 frame/s rate is achievable. The increased amount of recorded data raises the necessity of efficient automatic analysis methods. A set of images recorded on frog skeletal muscle cells after caffeine treatment was used to test the described methods.We present a collection of methods useful for the automatic analysis of high speed line scan and X-Y recordings, mostly based on stationary wavelet transform (SWT). Our one-dimensional SWT based ember-detection method was adapted to detect sparks on high-speed line scan recordings. The centerline of detected sparks is automatically marked on each scan line to assess the spatial and temporal characteristics of the spark. 3.66% of all sparks (n=23704) in the test image set had complex spatial properties revealed by high-speed imaging. Amplitude and full width at half maximum (FWHM) is determined for each scan line to calculate the amount of calcium released (signal mass).On X-Y image series two-dimensional SWT was used to denoise the images and to detect sparks on each frame. The orientation of the skeletal muscle cell and the position of sarcomeres are determined using fast Fourier-transform on the stationary components of the image series. The position of the center for each spark is determined relatively to the sarcomeres. FWHM is calculated in the axes perpendicular (FWHMX) and parallel (FWHMY) to the sarcomeres. For the test dataset (n=22426), FWHMX/FWHMY is 1.22.The above-described methods enable us to automatically analyze high-speed confocal images and to reveal some of their properties which were hidden on conventional confocal images.

Signal mass and Ca2+ kinetics in local calcium events: a modeling study

We use a detailed modeling formalism based on numerical simulations of local calcium release events where the blurring of the image, the presence of diffusional barriers provided by large organelles situated close to the release site, as well as the variable position of the scan line with respect to the release site are taken into consideration. We have investigated the effect of the fluorescence noise fluctuations on the accuracy in computing the signal mass from linescan recordings and obtained a quantitative description of both the signal mass and the local increase in the free Ca(2+) level as a function of the release current, the release duration and the orientation of the scan line, for three different levels of noise magnitudes. The model could provide a very good fit to a wide set of available experimental data regarding the signal mass of puffs visualized by fluorescence microscopy in the Xenopus oocyte loaded with 40 μM Oregon Green-1 in the absence of the calcium chelator EGTA. Numerical simulations also predict the amplitude and the kinetics of calcium signals evolving in the absence of the indicator, and indicate that sub-maximal activation of IP(3) receptors could produce in average levels of about 2 μM and 0.4 μM free Ca(2+) close to a release site located in the animal or in the vegetal hemisphere, respectively, whereas the maximal levels reached in more rare events could be 11 μM and 4 μM, respectively.

Coherent calcium puff signals driven by intracellular noises

In many cell types, intracellular calcium is released from internal stores through calcium release channels which are distributed in clusters with a few tens of channels. Localized calcium release events, i.e. Ca2 + puffs, are subjected to stochastic channel dynamics and fluctuations of environmental calcium. Driven by the internal channel noise or external calcium noise, the localized calcium puffs show a coherence resonance phenomenon at weak stimulus. Our study indicates that coherent calcium puffs with an enhanced periodicity can be achieved with external calcium noise more easily than with internal channel noise.

Theory and applications of geometric scaling of localized calcium release events

Geometric measures of localized calcium release (LCR) events have been used to understand their biophysical basis. We found power law scaling between three such metrics-maximum amplitude (MA), mass above half-maximum amplitude (MHM), and area at half-maximum amplitude (AHM). In an effort to understand this scaling a minimal analytic model was employed to simulate LCR events recorded by confocal line scan. The distribution of logMHM as a function of logAHM, pMHM(pAHM), was dependent on model parameters such as channel open time, current size, line scan offset, and apparent diffusion coefficient. The distribution of log[MHM/AHM] as a function of logMA, p[MHM/AHM](pMA), was invariant, reflecting the gross geometry of the LCR event. The findings of the model were applied to real LCR line scan data from rabbit portal vein myocytes, rat cerebral artery myocytes, and guinea pig fundus knurled cells. pMHM(pAHM) could be used to distinguish two populations of LCR events in portal vein, even at the scale of "calcium noise," and to calculate the relative current of the two. The relative current was 2. pMHM(pAHM) could also be used to study pharmacological effects. The pMHM(pAHM) distribution of knurled cell LCR events was markedly contracted by ryanodine, suggesting a reduction in channel open time. The p[MHM/AHM](pMA) distributions were invariant across all cell types and were consistent with the model, underlying the common physical basis of their geometry. The geometric scaling of LCR events demonstrated here may help with their mechanistic characterization.

Occurrence of Spontaneous Sparks in Ventricular Myocytes From Junctional and Non-junctional Ryr Clusters

In isolated rabbit ventricular myocytes, we found a significant number of ryanodine receptor (RyR) clusters that are not associated with the sarcolemma (non-junctional RyRs). The contribution of non-junctional RyR clusters to calcium transients is unclear. Here, we investigated if these non-junctional RyRs are able to produce spontaneous local calcium release events (sparks), and compared the probability of non-junctional versus junctional sparks. We imaged spontaneous sparks in cells loaded with fluo-4 and bathed in Tyrode solution with dextran (Molecular Weight: 10 kDa) linked to Texas Red dye. We evoked spontaneous sparks using field stimulation in the presence of 1 μM isoproterenol and 4 mM calcium. After 5 stimuli applied with a frequency of 0.5 Hz, we simultaneously imaged the sarcolemma and spontaneous sparks using a line scan confocal microscope (Biorad MRC-1024). Furthermore, a 3D image stack of the Texas Red associated signal was acquired to identify the sarcolemma including the transverse tubular system. We classified sparks as non-junctional if their distance to the sarcolemma is larger than 1 μm. All other sparks were assumed to be junctional. In measurements on 12 isolated cells, 38 sparks (51%) were identified as non-junctional, 36 (49%) as junctional. Our measurements clearly demonstrate that non-junctional RyR clusters are able to release calcium and produce spontaneous sparks. We expect that our approach for distinguishing between non-junctional and junctional sparks underestimates the number of non-junctional sparks. If this is true, the probabilities of the types of sparks are similar to probabilities of the two types of RyR clusters identified in related immunolabeling and microscopic studies. This would suggests that spark generation probability of RyR clusters does not depend on their type.

Altered expression of triadin 95 causes parallel changes in localized Ca2+ release events and global Ca2+ signals in skeletal muscle cells in culture

The 95 kDa triadin (Trisk 95), an integral protein of the sarcoplasmic reticular membrane in skeletal muscle, interacts with both the ryanodine receptor (RyR) and calsequestrin. While its role in the regulation of calcium homeostasis has been extensively studied, data are not available on whether the overexpression or the interference with the expression of Trisk 95 would affect calcium sparks the localized events of calcium release (LCRE). In the present study LCRE and calcium transients were studied using laser scanning confocal microscopy on C2C12 cells and on primary cultures of skeletal muscle. Liposome- or adenovirus-mediated Trisk 95 overexpression and shRNA interference with triadin translation were used to modify the level of the protein. Stable overexpression in C2C12 cells significantly decreased the amplitude and frequency of calcium sparks, and the frequency of embers. In line with these observations, depolarization-evoked calcium transients were also suppressed. Similarly, adenoviral transfection of Trisk 95 into cultured mouse skeletal muscle cells significantly decreased both the frequency and amplitude of spontaneous global calcium transients. Inhibition of endogenous triadin expression by RNA interference caused opposite effects. Primary cultures of rat skeletal muscle cells expressing endogenous Trisk 95 readily generated spontaneous calcium transients but rarely produced calcium sparks. Their transfection with specific shRNA sequence significantly reduced the triadin-specific immunoreactivity. Functional experiments on these cells revealed that while caffeine-evoked calcium transients were reduced, LCRE appeared with higher frequency. These results suggest that Trisk 95 negatively regulates RyR function by suppressing localized calcium release events and global calcium signals in cultured muscle cells.

Effects of Intermittent Femoral Nerve Injections of Bupivacaine, Levobupivacaine, and Ropivacaine on Mitochondrial Energy Metabolism and Intracellular Calcium Homeostasis in Rat Psoas Muscle

Long-acting local anesthetics cause muscle damage. Moreover, long-acting local anesthetics act as uncoupler of oxidative phosphorylation in isolated mitochondria and enhance sarcoplasmic reticulum Ca(2+) release. The aim of the study was to evaluate effects of perineural injections of local anesthetics on mitochondrial energetic metabolism and intracellular calcium homeostasis in vivo. Femoral nerve block catheters were inserted in adult male Wistar rats. Rats were randomized and received seven injections (1 ml/kg) of bupivacaine, levobupivacaine, ropivacaine, or isotonic saline at 8-h intervals. Rats were killed 8 h after the last injection. Psoas muscle was quickly dissected from next to the femoral nerve. Local anesthetic concentrations in muscle were determined. Oxidative capacity was measured in saponin-skinned fibers. Oxygen consumption rates were measured, and mitochondrial adenosine triphosphate synthesis rate was determined. Enzymatic activities of mitochondrial respiratory chain complexes were evaluated. Local calcium release events (calcium sparks) were analyzed as well as sarcoplasmic reticulum calcium content in saponin-skinned fibers. Eight hours after the last injection, psoas muscle concentration of local anesthetics was less than 0.3 microg/g tissue. Adenosine triphosphate synthesis and adenosine triphosphate-to-oxygen ratio were significantly decreased in the muscle of rats treated with local anesthetics. A global decrease (around 50%) in all of the enzyme activities of the respiratory chain was observed. Levobupivacaine increased the amplitude and frequency of the calcium sparks, whereas lower sarcoplasmic reticulum calcium content was shown. Bupivacaine, levobupivacaine, and ropivacaine injected via femoral nerve block catheters induce a deleterious effect in mitochondrial energy, whereas only levobupivacaine disturbs calcium homeostasis.

Localised calcium release events in cells from the muscle of guinea-pig gastric fundus.

After enzymatic dispersion of the muscle of the guinea-pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non-propagating) intracellular calcium ([Ca(2+)](i)) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio-temporal characteristics (full width and full duration at half-maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell-wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium-free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2-APB (100 microm), ryanodine (10 or 50 microm) or U-73122 (1 microm). 2-APB (100 microm), xestospongin C (XeC, 10 microm) or U-73122 (1 microm) blocked both spontaneous localized calcium release and localized release stimulated by 10 microm carbachol. Ryanodine (50 microm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.

Effects of ryanodine on calcium sparks in cut twitch fibres of Rana temporaria.

1. Localized calcium release events (calcium sparks) were studied in voltage-clamped cut twitch fibres of Rana temporaria. 2. A histogram of thousands of spontaneous sparks displayed a monotonically decreasing amplitude distribution from the low to the high limit of > 7 DeltaF/F(0) units. 3. Several effects of low micromolar concentrations of ryanodine (0.4-2 microM) on spontaneous sparks, reproducing the agent's effects on single ryanodine receptor channel current in bilayers, were observed collectively for the first time in live fibres, namely (a) increases in spark frequency followed by (b) conversions of sparks into steady glows lasting tens of seconds, (c) occasional interruptions of the glows by brief gaps of darkness, and (d) abolition of sparks at the locations of the glows. The glow could reflect the incessant Ca(2+) flux through a single (or a few) calcium release channel locked in the semi-open state, which was allowed to make occasional transitions to the closed state but not to the fully open state. 4. Higher concentrations of ryanodine (> or = 20 microM) suppressed the spontaneous sparks effectively and permanently, presumably by deactivating the ryanodine receptors. 5. Depolarization-evoked sparks elicited with small pulses had higher frequencies and larger amplitudes than spontaneous sparks and were abolished by both concentrations of ryanodine. 6. With 1-2 microM ryanodine, however, a uniform non-sparking calcium release persisted during the pulse, with the globally averaged increase in fluorescence intensity being about half that of the control. A possible origin of this non-sparking release may be related to the structural coupling between the voltage sensors and the ryanodine receptors that can exist only in live fibres but not in the bilayer preparation.

Effects of Imperatoxin A on Local Sarcoplasmic Reticulum Ca 2+ Release in Frog Skeletal Muscle

We have investigated the effects of imperatoxin A (IpTx a) on local calcium release events in permeabilized frog skeletal muscle fibers, using laser scanning confocal microscopy in linescan mode. IpTx a induced the appearance of Ca 2+ release events from the sarcoplasmic reticulum that are ∼2 s and have a smaller amplitude (31 ± 2%) than the “Ca 2+ sparks” normally seen in the absence of toxin. The frequency of occurrence of long-duration imperatoxin-induced Ca 2+ release events increased in proportion to IpTx a concentrations ranging from 10 nM to 50 nM. The mean duration of imperatoxin-induced events in muscle fibers was independent of toxin concentration and agreed closely with the channel open time in experiments on isolated frog ryanodine receptors (RyRs) reconstituted in planar lipid bilayer, where IpTx a induced opening of single Ca 2+ release channels to prolonged subconductance states. These results suggest involvement of a single molecule of IpTx a in the activation of a single Ca 2+ release channel to produce a long-duration event. Assuming the ratio of full conductance to subconductance to be the same in the fibers as in bilayer, the amplitude of a spark relative to the long event indicates involvement of at most four RyR Ca 2+ release channels in the production of short-duration Ca 2+ sparks.