Local Calcium Release Research Articles

Autonomic Stimulation Modulates Action Potential Firing Rate in Cardiac Pacemaker Cells via Synchronization of Local Calcium Pumping and Release

A modern view on regulation of cardiac pacemaker cell function postulates that local calcium releases (LCRs) contribute to diastolic depolarization via electrogenic sodium/calcium exchanger. An unresolved problem, however, remains: how intrinsically stochastic and heterogeneously distributed release channels (RyR) generate a strong, synchronized ensemble LCR signal and how this signal is effectively regulated by autonomic system to insure pacemaker rate flexibility.We measured calcium dynamics by a high-speed camera in isolated, single rabbit sinoatrial node cells (SANC) and assessed the kinetics of local calcium-pumping synchronization by examining the distributions of time constants (τ) of calcium-transient decay among cell neighborhoods at baseline and during stimulation of either beta-adrenergic receptors or cholinergic receptors.Beta-adrenergic receptor stimulation (isoproterenol) not only decreased cycle length (CL) and average τ vs. baseline, but also decreased the standard deviation (SD) of all τ's across all neighborhoods, suggesting a shift into a less heterogeneous, i.e. more synchronized calcium pumping throughout SANC. Conversely, cholinergic receptor stimulation (carbachol) not only increased CL and average τ vs. baseline, but also increased the local SD(τ), suggesting a shift into a more heterogeneous, i.e. less synchronized calcium pumping within SANC. Furthermore, on a beat-to-beat basis, the relationship between either τ or SD(τ) and CL was linear under both baseline conditions and autonomic stimulation.Conclusions: The degree of heterogeneity of local calcium pumping is a new universal factor that affects the CL and insures effective rate and rhythm regulation of the coupled-clock pacemaker system via autonomic modulation. More synchronized and faster calcium pumping is presumably achieved via phospholamban phosphorylation and allows cell neighborhoods to reach the calcium release threshold quicker and more synchronously, thereby synchronizing LCRs and amplifying their ensemble diastolic signal, accelerating pacemaker rate.

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.

Neuronal regeneration in C. elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation.

Regulated calcium signals play conserved instructive roles in neuronal repair, but how localized calcium stores are differentially mobilized, or might be directly manipulated, to stimulate regeneration within native contexts is poorly understood. We find here that localized calcium release from the endoplasmic reticulum via ryanodine receptor (RyR) channels is critical in stimulating initial regeneration following traumatic cellular damage in vivo. Using laser axotomy of single neurons in Caenorhabditis elegans, we find that mutation of unc-68/RyR greatly impedes both outgrowth and guidance of the regenerating neuron. Performing extended in vivo calcium imaging, we measure subcellular calcium signals within the immediate vicinity of the regenerating axon end that are sustained for hours following axotomy and completely eliminated within unc-68/RyR mutants. Finally, using a novel optogenetic approach to periodically photo-stimulate the axotomized neuron, we can enhance its regeneration. The enhanced outgrowth depends on both amplitude and temporal pattern of excitation and can be blocked by disruption of UNC-68/RyR. This demonstrates the exciting potential of emerging optogenetic technology to beneficially manipulate cell physiology in the context of neuronal regeneration and indicates a link to the underlying cellular calcium signal. Taken as a whole, our findings define a specific localized calcium signal mediated by RyR channel activity that stimulates regenerative outgrowth, which may be dynamically manipulated for beneficial neurotherapeutic effects.

Regulation of metabolism in individual mitochondria during excitation–contraction coupling

The heart is an excitable organ that undergoes spontaneous force generation and relaxation cycles driven by excitation–contraction (EC) coupling. A fraction of the oscillating cytosolic Ca2+ during each heartbeat is taken up by mitochondria to stimulate mitochondrial metabolism, the major source of energy in the heart. Whether the mitochondrial metabolism is regulated individually during EC coupling and whether this heterogeneous regulation bears any physiological or pathological relevance have not been studied. Here, we developed a novel approach to determine the regulation of individual mitochondrial metabolism during cardiac EC coupling. Through monitoring superoxide flashes, which are stochastic and bursting superoxide production events arising from increased metabolism in individual mitochondria, we found that EC coupling stimulated the metabolism in individual mitochondria as indicated by significantly increased superoxide flash activity during electrical stimulation of the cultured intact myocytes or perfused heart. Mechanistically, cytosolic calcium transients promoted individual mitochondria to take up calcium via mitochondrial calcium uniporter, which subsequently triggered transient opening of the permeability transition pore and stimulated metabolism and bursting superoxide flash in that mitochondrion. The bursting superoxide, in turn, promoted local calcium release. In the early stage of heart failure, EC coupling regulation of superoxide flashes was compromised. This study highlights the heterogeneity in the regulation of cardiac mitochondrial metabolism, which may contribute to local redox signaling.

Excitability in a stochastic differential equation model for calcium puffs

Calcium dynamics are essential to a multitude of cellular processes. For many cell types, localized discharges of calcium through small clusters of intracellular channels are building blocks for all spatially extended calcium signals. Because of the large noise amplitude, the validity of noise-approximating model equations for this system has been questioned. Here we revisit the master equations for local calcium release, examine the multiple scales of calcium concentrations in the cluster domain, and derive adapted stochastic differential equations. We show by comparison of discrete and continuous trajectories that the Langevin equations can be made consistent with the master equations even for very small channel numbers. In its deterministic limit, the model reveals that excitability, a dynamical phenomenon observed in many natural systems, is at the core of calcium puffs. The model also predicts a bifurcation from transient to sustained release which may link local and global calcium signals in cells.

Frequency and Relative Prevalence of Calcium Blips and Puffs in a Model of Small IP3R Clusters

In this work, we model the local calcium release from clusters with a few inositol 1,4,5-trisphosphate receptor (IP3R) channels, focusing on the stochastic process in which an open channel either triggers other channels to open (as a puff) or fails to cause any channel to open (as a blip). We show that there are linear relations for the interevent interval (including blips and puffs) and the first event latency against the inverse cluster size. However, nonlinearity is found for the interpuff interval and the first puff latency against the inverse cluster size. Furthermore, the simulations indicate that the blip fraction among all release events and the blip frequency are increasing with larger basal [Ca2+], with blips in turn giving a growing contribution to basal [Ca2+]. This result suggests that blips are not just lapses to trigger puffs, but they may also possess a biological function to contribute to the initiation of calcium waves by a preceding increase of basal [Ca2+] in cells that have small IP3R clusters.

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.

Major role of ryanodine type 2 receptors in global and local intracellular calcium release in arterial smooth muscle (1067.7)

Ryanodine receptors (RyR) are calcium‐sensitive ion channels in the sarcoplasmic reticulum (SR) membrane, and are important effectors in arterial smooth muscle excitation‐contraction (EC) coupling. While three different RyR isoforms are expressed in arterial smooth muscle cells (SMCs), the relative contribution of each RyR isoform to vascular function remains unresolved. We generated tamoxifen‐inducible SMC specific knock‐out mice of RyR2 to test the hypothesis of RyR2 contributing in arterial EC coupling, vasoregulation, and systemic blood pressure. Myography and videomicroscopy of isolated arteries, calcium imaging and patch clamp current recordings on isolated SMCs, and radiotelemetric blood pressure measurements were used. Targeted deletion of the gene for RyR2 resulted in a loss in functional coupling of calcium sparks to associated BK potassium channel activation, and increased arterial tone and systemic blood pressure. Aortas, femoral, cerebral and mesenteric arteries from RyR2‐deficient mice showed no contraction in response to RyR activation by 10 mmol/L caffeine. Our data demonstrate that RyR2 are key elements in Cav1.2 channel‐induced EC coupling in large and small arteries. Furthermore, RyR2, by activating BK channels, are important molecular SR calcium release components in translating local calcium signals (Ca2+‐sparks) to vasoregulation and arterial blood pressure.Grant Funding Source: Supported by the Deutsche Forschungsgemeinschaft (DFG)

Investigation of Arrhythmogenic Calcium Events by Initiating Local Calcium Release in Cardiomyocytes

In cardiac muscle the sarcoplasmic reticulum (SR) contains the Ca2+ that is released during excitation-contraction coupling to initiate cross-bridge cycling, sarcomere shortening and force generation. Under physiological conditions SR Ca2+ uptake by the SR/ER Ca2+ ATPase is balanced by Ca2+ “leak” out of the SR through the SR Ca2+ release channels (ryanodine receptors, RyR2). Under diverse conditions SR Ca2+ overload can develop (i.e. elevation of [Ca2+]SR) and this is associated with an increase in the open probability of the RyR2s which leads to SR Ca2+ instability.

Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel

Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF hand containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity to Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion.

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.

Mechanical unloading reverses transverse tubule remodelling and normalises local calcium-induced calcium release in a rodent model of heart failure

Calcium-induced calcium release (CICR) is crucial for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for calcium release (L-type calcium channel, dihydropyridine receptors) and the sarcoplasmic reticulum calcium-release channels (ryanodine receptors). Transverse tubule disruption occurs early in heart failure. Clinical studies of left ventricular assist devices in heart failure indicate that mechanical unloading induces reverse remodelling. We hypothesise that unloading of failing hearts normalises t-tubule structure and improves CICR.Heart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). Heart failure reduced the t-tubule density as measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in heart failure was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type calcium current density, measured with whole-cell patch clamping, was reduced in heart failure but unaffected by unloading. The variance of the time-to-peak of the calcium transient, an index of CICR dyssynchrony, was increased in heart failure and normalised by unloading. The increased calcium spark frequency observed in heart failure was reduced in HF-UN. These results could be explained by the recoupling of orphaned ryanodine receptors in heart failure, as indicated by immunofluorescence.Our data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR. FundingBritish Heart Foundation.

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.

Refractoriness of Calcium Release Units in Rat Cardiac Myocytes

Calcium release units (CRUs) in cardiac myocytes contain a large number of ryanodine receptors (RyRs), but it seems that only a small fraction of RyRs is being activated during a single calcium release event. Repetitive activity of CRUs is not well understood from the point of refractoriness. We used direct measurement of Ca-spikes activated by calcium current (ICa) to unveil the refractoriness of CRU firing from the properties and occurrence of calcium spikes under control conditions and in the presence of the DHPR activator FPL.ICa was activated by voltage pulses from 50 to 0 mV under whole-cell patch-clamp. Ca-spikes were measured by confocal microscopy using Ca2+ indicators (Fluo-3 and Oregon Green BAPTA-5N) in the presence of EGTA. A large majority of CRUs responded to stimulation by a single Ca-spike, while 13.4 % produced two subsequent (twin) Ca-spikes. Early Ca-spikes (single spikes and the first of twin Ca-spikes) had similar latencies, while the second Ca-spikes were significantly delayed. Amplitude distribution of the early Ca-spikes consisted of four quantal levels with equal amplitudes and binomially distributed frequency of occurrence, supporting the hypothesis that different Ca-spike amplitudes are due to a different number of independent quantal levels, likely individual RYR openings. The probability of occurrence of second Ca-spikes was inversely proportional to the quantal size of the early Ca-spike. In the presence of FPL, the probability of occurrence of twin spikes was decreased relative to control, despite persistent activation of DHPR receptors.We conclude that refractoriness of local calcium release does not result from Ca-dependent DHPR inactivation, but rather it is caused by inactivation of the release unit due to local depletion of the SR.Supported by APVV-0721-10, APVV-0441-09, VEGA 2/0203/11, VEGA 2/0917/11, and VEGA 2/0190/10.

Voltage Dependence of Calcium Spike Characteristics in Rat Cardiac Myocytes

Integral calcium release fluxes in mammalian cardiac myocytes increase in amplitude not only due to the increased recruitment of individual release sites but also due to their increased synchronization (1). Here we characterize the voltage dependence of synchronization of individual release sites at high temporal resolution. Calcium spikes and calcium currents elicited in isolated rat ventricular myocytes (2) by 80 ms voltage pulses from −50 to −40 - +50 mV were recorded using Leica TCS AOBS confocal microscope (2 kHz line scan mode) and Axopatch 200B patch-clamp amplifier, respectively. Calcium spikes were identified and analyzed by a new Matlab-based software that highly increased the productivity and reliability of approximation of calcium spikes by the kinetic model of local calcium release (2). Calcium spikes were described by their amplitude, onset latency, time-to-peak, FDHM, time constants of activation and termination, and fractional probability of activation. The synchrony of calcium spikes increased with depolarization and could be fully explained by the voltage dependence of their onset latency that reached a lower limit of about 4 ms at depolarizations between 0 and 40 mV. The rate of calcium spike activation after its onset was independent of the applied voltage or of calcium current amplitude, while the probability of calcium spike activation was related to the voltage dependence of the integral of calcium current. We conclude that both the synchrony of local calcium release activation and recruitment of individual release sites are fully controlled by the activation probability of DHPR calcium channels and by the DHPR-RYR coupling fidelity.Supported by APVV-0721-10, VEGA 2/0203/11, VEGA 2/0917/11.1. Song LS, Sham JS, Stern MD, Lakatta EG, Cheng H. J Physiol. 512:677-91, 1998.2. Zahradnikova A Jr, Polakova E, Zahradnik I, Zahradnikova A. J Physiol. 578:677-91, 2007.

Detection, properties, and frequency of local calcium release from the sarcoplasmic reticulum in teleost cardiomyocytes.

Calcium release from the sarcoplasmic reticulum (SR) plays a central role in the regulation of cardiac contraction and rhythm in mammals and humans but its role is controversial in teleosts. Since the zebrafish is an emerging model for studies of cardiovascular function and regeneration we here sought to determine if basic features of SR calcium release are phylogenetically conserved. Confocal calcium imaging was used to detect spontaneous calcium release (calcium sparks and waves) from the SR. Calcium sparks were detected in 16 of 38 trout atrial myocytes and 6 of 15 ventricular cells. The spark amplitude was 1.45±0.03 times the baseline fluorescence and the time to half maximal decay of sparks was 27±3 ms. Spark frequency was 0.88 sparks µm−1 min−1 while calcium waves were 8.5 times less frequent. Inhibition of SR calcium uptake reduced the calcium transient (F/F0) from 1.77±0.17 to 1.12±0.18 (p = 0.002) and abolished calcium sparks and waves. Moreover, elevation of extracellular calcium from 2 to 10 mM promoted early and delayed afterdepolarizations (from 0.6±0.3 min−1 to 8.1±2.0 min−1, p = 0.001), demonstrating the ability of SR calcium release to induce afterdepolarizations in the trout heart. Calcium sparks of similar width and duration were also observed in zebrafish ventricular myocytes. In conclusion, this is the first study to consistently report calcium sparks in teleosts and demonstrate that the basic features of calcium release through the ryanodine receptor are conserved, suggesting that teleost cardiac myocytes is a relevant model to study the functional impact of abnormal SR function.

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.

Challenging quantal calcium signaling in cardiac myocytes

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

Under pressure - Kvchannels and myogenic control of cerebral blood flow

Under pressure - K_vchannels and myogenic control of cerebral blood flow