Calcium-induced Release Research Articles

Up-regulation of ryanodine receptor expression increases the calcium-induced calcium release and spontaneous calcium signals in cerebral arteries from hindlimb unloaded rats

Microgravity induces a redistribution of blood volume. Consequently, astronauts' body pressure is modified so that the upright blood pressure gradient is abolished, thereby inducing a modification in cerebral blood pressure. This effect is mimicked in the hindlimb unloaded rat model. After a duration of 8 days of unloading, Ca2+ signals activated by depolarization and inositol-1,4,5-trisphosphate intracellular release were increased in cerebral arteries. In the presence of ryanodine and thapsigargin, the depolarization-induced Ca2+ signals remained increased in hindlimb suspended animals, indicating that Ca2+ influx and Ca2+-induced Ca2+ release mechanism were both increased. Spontaneous Ca2+ waves and localized Ca2+ events were also investigated. Increases in both amplitude and frequency of spontaneous Ca2+ waves were measured in hindlimb suspension conditions. After pharmacological segregation of Ca2+ sparks and Ca2+ sparklets, their kinetic parameters were characterized. Hindlimb suspension induced an increase in the frequencies of both Ca2+ localized events, suggesting an increase of excitability. Labeling with bodipy compounds suggested that voltage-dependent Ca2+ channels and ryanodine receptor expressions were increased. Finally, the expression of the ryanodine receptor subtype 1 (RyR1) was increased in hindlimb unloading conditions. Taken together, these results suggest that RyR1 expression and voltage-dependent Ca2+ channels activity are the focal points of the regulation of Ca2+ signals activated by vasoconstriction in rat cerebral arteries with an increase of the voltage-dependent Ca2+ influx.

Life and death of a cardiac calcium spark

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition-a phenomenon we dub "spark metastability." Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.

Life and death of a cardiac calcium spark

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.

Actein induces calcium release in human breast cancer cells

BackgroundThe triterpene glycoside actein from the herb black cohosh preferentially inhibits the growth of breast cancer cells and activates the ER stress response. The ER IP3 receptor and Na,K-ATPase form a signaling microdomain. Since actein is lipophilic, its action may be limited by bioavailability. PurposeTo develop actein to prevent and treat cancer, we examined the primary targets and combinations with chemotherapy agents, as well as the ability of nanoparticles to enhance the activity. Materials and methodsTo reveal signaling pathways, we treated human breast and colon cancer, as well as 293T and 293T (NF-κB), cells with actein, and measured effects using the MTT, luciferase promoter, Western blot and histology assays. To assess effects on calcium release, we preloaded cells with the calcium sensitive dye Fura-2. To enhance bioavailability, we conjugated actein to nanoparticle liposomes. ResultsActein strongly inhibited the growth of human breast cancer cells and induced a dose dependent release of calcium into the cytoplasm. The ER IP3 receptor antagonist heparin blocked this release, indicating that the receptor is required for activity. Heparin partially blocked the growth inhibitory effect, while the MEK inhibitor U0126 enhanced it. Consistent with this, actein synergized with the ER mobilizer thapsigargin. Further, actein preferentially inhibited the growth of 293T (NF-κB) cells. Nanoparticle liposomes increased the growth inhibitory activity of actein. ConclusionsActein alters the activity of the ER IP3 receptor and Na,K-ATPase, induces calcium release and modulates the NF-κB and MEK pathways and may be worthwhile to explore to prevent and treat breast cancer.

Mitochondrial Hyperfusion during Oxidative Stress Is Coupled to a Dysregulation in Calcium Handling within a C2C12 Cell Model

Atrial Fibrillation is the most common sustained cardiac arrhythmia worldwide harming millions of people every year. Atrial Fibrillation (AF) abruptly induces rapid conduction between atrial myocytes which is associated with oxidative stress and abnormal calcium handling. Unfortunately this new equilibrium promotes perpetuation of the arrhythmia. Recently, in addition to being the major source of oxidative stress within cells, mitochondria have been observed to fuse, forming mitochondrial networks and attach to intracellular calcium stores in response to cellular stress. We sought to identify a potential role for rapid stimulation, oxidative stress and mitochondrial hyperfusion in acute changes to myocyte calcium handling. In addition we hoped to link altered calcium handling to increased sarcoplasmic reticulum (SR)-mitochondrial contacts, the so-called mitochondrial associated membrane (MAM). We selected the C2C12 murine myotube model as it has previously been successfully used to investigate mitochondrial dynamics and has a myofibrillar system similar to atrial myocytes. We observed that rapid stimulation of C2C12 cells resulted in mitochondrial hyperfusion and increased mitochondrial colocalisation with calcium stores. Inhibition of mitochondrial fission by transfection of mutant DRP1K38E resulted in similar effects on mitochondrial fusion, SR colocalisation and altered calcium handling. Interestingly the effects of ‘forced fusion’ were reversed by co-incubation with the reducing agent N-Acetyl cysteine (NAC). Subsequently we demonstrated that oxidative stress resulted in similar reversible increases in mitochondrial fusion, SR-colocalisation and altered calcium handling. Finally, we believe we have identified that myocyte calcium handling is reliant on baseline levels of reactive oxygen species as co-incubation with NAC both reversed and retarded myocyte response to caffeine induced calcium release and re-uptake. Based on these results we conclude that the coordinate regulation of mitochondrial fusion and MAM contacts may form a point source for stress-induced arrhythmogenesis. We believe that the MAM merits further investigation as a therapeutic target in AF-induced remodelling.

Ryanodine Receptors Selectively Interact with L Type Calcium Channels in Mouse Taste Cells

IntroductionWe reported that ryanodine receptors are expressed in two different types of mammalian peripheral taste receptor cells: Type II and Type III cells. Type II cells lack voltage-gated calcium channels (VGCCs) and chemical synapses. In these cells, ryanodine receptors contribute to the taste-evoked calcium signals that are initiated by opening inositol trisphosphate receptors located on internal calcium stores. In Type III cells that do have VGCCs and chemical synapses, ryanodine receptors contribute to the depolarization-dependent calcium influx.Methodology/Principal FindingsThe goal of this study was to establish if there was selectivity in the type of VGCC that is associated with the ryanodine receptor in the Type III taste cells or if the ryanodine receptor opens irrespective of the calcium channels involved. We also wished to determine if the ryanodine receptors and VGCCs require a physical linkage to interact or are simply functionally associated with each other. Using calcium imaging and pharmacological inhibitors, we found that ryanodine receptors are selectively associated with L type VGCCs but likely not through a physical linkage.Conclusions/SignificanceTaste cells are able to undergo calcium induced calcium release through ryanodine receptors to increase the initial calcium influx signal and provide a larger calcium response than would otherwise occur when L type channels are activated in Type III taste cells.

Murine Muscle Engineered from Dermal Precursors: An In Vitro Model for Skeletal Muscle Generation, Degeneration, and Fatty Infiltration

Skeletal muscle can be engineered by converting dermal precursors into muscle progenitors and differentiated myocytes. However, the efficiency of muscle development remains relatively low and it is currently unclear if this is due to poor characterization of the myogenic precursors, the protocols used for cell differentiation, or a combination of both. In this study, we characterized myogenic precursors present in murine dermospheres, and evaluated mature myotubes grown in a novel three-dimensional culture system. After 5-7 days of differentiation, we observed isolated, twitching myotubes followed by spontaneous contractions of the entire tissue-engineered muscle construct on an extracellular matrix (ECM). In vitro engineered myofibers expressed canonical muscle markers and exhibited a skeletal (not cardiac) muscle ultrastructure, with numerous striations and the presence of aligned, enlarged mitochondria, intertwined with sarcoplasmic reticula (SR). Engineered myofibers exhibited Na(+)- and Ca(2+)-dependent inward currents upon acetylcholine (ACh) stimulation and tetrodotoxin-sensitive spontaneous action potentials. Moreover, ACh, nicotine, and caffeine elicited cytosolic Ca(2+) transients; fiber contractions coupled to these Ca(2+) transients suggest that Ca(2+) entry is activating calcium-induced calcium release from the SR. Blockade by d-tubocurarine of ACh-elicited inward currents and Ca(2+) transients suggests nicotinic receptor involvement. Interestingly, after 1 month, engineered muscle constructs showed progressive degradation of the myofibers concomitant with fatty infiltration, paralleling the natural course of muscular degeneration. We conclude that mature myofibers may be differentiated on the ECM from myogenic precursor cells present in murine dermospheres, in an in vitro system that mimics some characteristics found in aging and muscular degeneration.

Recruitment of the intracellular Ca2+ by ultrashort electric stimuli: The impact of pulse duration

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca2+ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca2+ imaging with Fura-2. In a Ca2+-free medium, 10-, 60-, and 300-ns stimuli evoked Ca2+ transients by mobilization of Ca2+ from the endoplasmic reticulum. With 2mM external Ca2+, the transients included both extra- and intracellular components. The recruitment of intracellular Ca2+ increased as the stimulus duration decreased. At the threshold of 200–300nM, the transients were amplified by calcium-induced calcium release. We conclude that nanosecond stimuli mimic Ca2+ signaling while bypassing the usual receptor- and channels-mediated cascades. The recruitment of the intracellular Ca2+ can be controlled by the duration of the stimulus.

Control of Sarcoplasmic Reticulum Ca2+ Release by Stochastic RyR Gating within a 3D Model of the Cardiac Dyad and Importance of Induction Decay for CICR Termination

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca2+ spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca2+, Mg2+, and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca2+ sparks, Ca2+ blinks, and Ca2+ spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca2+ dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca2+ dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca2+, as well as the time course of local Ca2+ gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca2+ spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca2+ spark initiation and subsequent Ca2+ spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca2+ in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca2+ spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca2+ flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca2+ sensitivities, Ca2+ spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.

Extinguishing the Sparks

The mechanical contraction of cardiac muscle is triggered by membrane depolarization through a process known as calcium-induced calcium-release (CICR). The structural basis of CICR is exquisite in its detail. At thousands of distinct locations throughout the myocyte, the junctional sarcoplasmic reticulum (JSR) membrane approaches to within ∼12 nm of the t-tubule membrane to form structures known as dyads. Dyads consist of a handful of L-type calcium (Ca2+) channels (LCCs) in the t-tubule membrane, a larger number of ryanodine-sensitive Ca2+-gated Ca2+-release channels (RyR2s) in the closely apposed JSR membrane, regulatory proteins that modify LCC and RyR2 function, and the fluid volume that separates them. Membrane depolarization leads to openings of LCCs, flux of Ca2+ into the dyadic space, Ca2+ binding to and opening of RyR2s, and release of Ca2+ from the JSR into the dyadic space. This Ca2+ then diffuses out of the dyad and binds to the mechanical machinery of the cell. The sum of these Ca2+ release events drives contraction of heart muscle.

Palmitate Diet-induced Loss of Cardiac Caveolin-3: A Novel Mechanism for Lipid-induced Contractile Dysfunction

Obesity is associated with an increased risk of cardiomyopathy, and mechanisms linking the underlying risk and dietary factors are not well understood. We tested the hypothesis that dietary intake of saturated fat increases the levels of sphingolipids, namely ceramide and sphingomyelin in cardiac cell membranes that disrupt caveolae, specialized membrane micro-domains and important for cellular signaling. C57BL/6 mice were fed two high-fat diets: palmitate diet (21% total fat, 47% is palmitate), and MCT diet (21% medium-chain triglycerides, no palmitate). We established that high-palmitate feeding for 12 weeks leads to 40% and 50% increases in ceramide and sphingomyelin, respectively, in cellular membranes. Concomitant with sphingolipid accumulation, we observed a 40% reduction in systolic contractile performance. To explore the relationship of increased sphingolipids with caveolins, we analyzed caveolin protein levels and intracellular localization in isolated cardiomyocytes. In normal cardiomyocytes, caveolin-1 and caveolin-3 co-localize at the plasma membrane and the T-tubule system. However, mice maintained on palmitate lost 80% of caveolin-3, mainly from the T-tubule system. Mice maintained on MCT diet had a 90% reduction in caveolin-1. These data show that caveolin isoforms are sensitive to the lipid environment. These data are further supported by similar findings in human cardiac tissue samples from non-obese, obese, non-obese cardiomyopathic, and obese cardiomyopathic patients. To further elucidate the contractile dysfunction associated with the loss of caveolin-3, we determined the localization of the ryanodine receptor and found lower expression and loss of the striated appearance of this protein. We suggest that palmitate-induced loss of caveolin-3 results in cardiac contractile dysfunction via a defect in calcium-induced calcium release.

Complex Actions of Ionomycin in Cultured Cerebellar Astrocytes Affecting Both Calcium-Induced Calcium Release and Store-Operated Calcium Entry

The polyether antibiotic ionomycin is a common research tool employed to raise cytosolic Ca(2+) in almost any cell type. Although initially thought to directly cause physicochemical translocation of extracellular Ca(2+) into the cytosol, a number of studies have demonstrated that the mechanism of action is likely to be more complex, involving modulation of intrinsic Ca(2+) signaling pathways. In the present study we assessed the effect of ionomycin on primary cultures of murine cerebellar astrocytes. Ionomycin concentrations ranging from 0.1 to 10μM triggered a biphasic increase in cytosolic Ca(2+), consisting of an initial peak and a subsequent sustained plateau. The response was dependent on concentration and exposure time. While the plateau phase was abolished in the absence of extracellular Ca(2+), the peak phase persisted. The peak amplitude could be lowered significantly by application of dantrolene, demonstrating involvement of Ca(2+)-induced Ca(2+)-release (CICR). The plateau phase was markedly reduced when store-operated Ca(2+)-entry (SOCE) was blocked with 2-aminoethoxydiphenyl borate. Our results show that ionomycin directly affects internal Ca(2+) stores in astrocytes, causing release of Ca(2+) into the cytosol, which in turn triggers further depletion of the stores through CICR and subsequently activates SOCE. This mechanistic action of ionomycin is important to keep in mind when employing it as a pharmacological tool.

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.

Termination of Calcium Sparks: An Emergent Property from Stochastic RyR Gating and Dyad Geometry

Many cells depend on ‘calcium-induced calcium release' (CICR), an inherently regenerative process due to the Ca2+-dependent gating of ryanodine receptors (RyRs) in the SR. Ca2+ sparks, as seen in muscle, reflect the concerted gating of groups of RyRs in specialized signalling domains in the junctions between the SR and surface membrane. However, the mechanism(s) responsible for the termination of regenerative CICR during the evolution of Ca2+ sparks remain uncertain. Rat cardiac RyR gating was recorded at physiological Ca2+, Mg2+ and ATP levels and incorporated into a 3D spatial model of the cardiac dyad which reproduced the time-course of Ca2+ sparks, Ca2+ blinks and Ca2+ spark restitution. Model CICR termination was robust, relatively insensitive to the number dyadic RyRs and automatic. This emergent behavior arose from the rapid development and dissolution of nanoscopic Ca2+ gradients within the dyad. These simulations show that CICR does not require intrinsic inactivation mechanisms for stability and cessation of regeneration arises from local control at the molecular scale via a process we call ‘induction decay'.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Non-Ligand Mobilization of Intracellular Free Calcium by Nanosecond Pulsed Electric Field (NsPEF)

NsPEF makes membrane and intracellular structures in living cells permeable to ions and small molecules thus can be used to modulate cell's signaling pathways and homeostasis. We utilized Fura-2 fast ratiometric recordings to measure changes of intracellular free calcium concentration ([Ca2+]i) in single CHO cells stimulated by 10, 60 and 300 ns PEF. 10 and 300 ns PEF were used for comparison with 60 ns PEF in regard to their potency to make plasma membrane (PM) and/or endoplasmic reticulum (ER) permeable to Ca2+ ions. Single 60 ns PEF evoked transient [Ca2+]i rise due to Ca2+ influx via PM and Ca2+ efflux from ER with thresholds ∼9 and ∼19 kV/cm, respectively. The amplitude of Ca2+ rise increased with nsPEF intensity linearly by 8-10nM per 1 kV/cm (PM) or 5-6 nM per 1 kV/cm when permeabilization of ER was assessed in Ca2+ free solution. This rate of change increased drastically (to 174nM per 1 kV/cm) when amplitude of Ca2+ rise reached 100-150nM. Depleting Ca2+ content of ER showed that such increase was a result of calcium induced calcium release (CICR) activation. Specific blockers 2-APB and dantrolene proved that the activation of IP3 receptors was responsible for physiological mechanism of CICR triggered by nsPEF. 10, 60 and 300 ns PEF had different efficiencies in regard to PM and ER permeabilization for Ca2+ ions. The thresholds of PM and ER permeabilization were indistinguishable for 10 ns pulse while for 60 ns pulse the ER threshold was twofold higher. 300 ns pulse had further reduced potency towards ER permeabilization in comparison to either 10 or 60 ns pulses. These results show that nsPEF can be effectively used to manipulate [Ca2+]i and activate intracellular signaling pathways bypassing PM receptors.Supported by R01CA125482; R01GM088303

In Situ Visualization of Mouse Skeletal Muscle Ryanodine Receptor by Cryo-Electron Tomography

Among three isoforms of mouse ryanodine receptors (RyRs), which share 70% similarities with their amino acid sequences, the type 1 skeletal muscle ryanodine receptor (RyR1) is the most extensively studied and known. Structurally, RyR1 is made up of four identical subunits. Each subunit has a molecular mass of approximately 500-600 kDa. This isoform contains at least two functional domains: a carboxyl-terminal hydrophobic domain or trans-membrane domain that contains the Ca2+ release channel pore, and a large amino-terminal domain or cytoplasmic domain that contains sites responsible for the channel regulation. Physiologically, RyR1 functions as a channel for calcium-induced calcium release (CICR) during excitation-contraction coupling (EC-coupling), in which Ca2+ release from the sarcoplasmic reticulum is followed by the subsequent muscle contraction. In central core disease, mutations in RyR1 may promote disruption of EC-coupling resulting in skeletal muscle weakness and lower limb deformities. The important role for RyR1 in this physiological function drives the need for high resolution and comprehensive structural information. Despite many structural studies on RyR1, however, the mechanism underlying this phenomenon remains enigmatic. Advances in techniques and approaches to observe a structure of RyR1 is important to address this question. In this study, we used immunogold for in situ visualization of mice RyR1 by electron tomography. To our knowledge, this is the first report on the application of electron tomography for in situ visualization of RyR1.

Co-Expression of TRIC-A and Cardiac Ryanodine Receptor affects Store-Overload Induced Calcium Release in HEK293 Cells

TRIC represents a novel class of trimeric intracellular cation channels located at the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) of multiple cell types. These channels regulate the permeability of K ions across the ER/SR and consequently the movement of Ca ions during excitation-contraction coupling. Previously we showed that genetic ablation of TRIC-A lead to compromised K-permeability and Ca release across the SR/ER membrane, supporting the hypothesis that TRIC could function as counter-ion channels that provide the flow of K ions into the SR during the acute phase of Ca release. In the absence of TRIC, overload of Ca inside the ER/SR causes instability of Ca storage and release, leading to stress-induced dysfunction of skeletal and cardiac muscles. Cardiomyocytes isolated from the wild type mice often show spontaneous Ca release during SR Ca overload, also called store-overload induced Ca release (SOICR) that is mediated by the type 2 ryanodine receptor (RyR2). The appearance of SOICR was significantly less frequent in the TRIC-A knockout cardiomyocytes. Biochemical studies revealed that the carboxyl-tail domain of TRIC-A could interact with the RyR channel, supporting the possibility that TRIC-A may directly regulate the Ca release activity. It has been shown that SOICR can be reconstituted in HEK293 cells expressing RyR2. We found that expression of TRIC-A in these RyR2-expressing HEK293 cells could prevent overload of Ca inside the ER and abolish SOICR generation. Together, our data suggest that functional interaction between TRIC-A and RyR can modulate the Ca release process from internal stores.

Modeling the Effect of Unitary Calcium Current on Neighboring Ryanodine Receptors during Calcium Induced Calcium Release

In resting cardiac cells, the open probability (Po) of single ryanodine receptors (RyRs) is very low and consequently little Ca2+ is released from the sarcoplasmic reticulum (SR). However, a stochastic RyR opening will cause diastolic local Ca2+ release from the SR that can activate neighboring closed RyRs. This inter-RyR Ca2+-induced Ca2+ release (CICR) may generate diastolic Ca2+ sparks. It is known that elevating SR Ca2+ load above normal levels dramatically increases spark frequency and increases the unitary RyR Ca2+ current. It is this current that acts on neighboring RyRs through CICR. We have developed a simple model based on experimental single-channel RyR Ca2+ sensitivity to understand how unitary RyR Ca2+ current may control CICR within a group of neighboring RyRs (a Ca2+ release unit, CRU). The model predicts how the current carried by an open RyR influences the activity of neighboring RyRs in a CRU. These predictions match published experimental single and clustered RyR channel results obtained in bilayer studies.

A Luminal Calcium Sensing Mutation of the Cardiac Ryanodine Receptor Diminishes Calcium Waves and Stress -Induced Ventricular Tachycardias in Calsequestrin Null Mice

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease. Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2) have been linked to CPVT. The sarcoplasmic reticulum calcium content and the sensitivity of RyR2 to luminal calcium are two important factors determining the propensity for store overload induced spontaneous calcium release, also known as store-overload induced calcium release (SOICR), and SOICR-evoked VTs. We recently found that residue E4872 of RyR2 is critical for luminal calcium sensing. In the present study, we determined whether the RyR2-E4872Q mutation is capable of rescuing the VT phenotype of the CASQ2 null mice. We bred E4872Q+/- mice with CASQ2 null mice to produce CASQ2-/- or CASQ2+/- mice with or without the RyR2-E4872Q mutation. CASQ2-/- and CASQ2+/- mice without the RyR2-E4872Q mutation showed long lasting stress-induced VTs, whereas CASQ2-/- / E4872Q+/- and CASQ2+/- / E4872Q +/- displayed little or no VTs. Thus, the RyR2-E4872Q mutation nearly completely protected the CASQ2 null mice from CPVT. We next studied the impact of the RyR2-E4872Q mutation on calcium handling in intact hearts using linescan confocal calcium imaging. The properties of calcium transients did not differ significantly between CASQ2-/- hearts with or without the RyR2-E4872Q mutation at resting conditions. However, upon elevation of external calcium from 2mM to 7mM or adding 100nM isoproterenol, frequent spontaneous calcium waves occurred in CASQ2-/- hearts without the RyR2-E4872Q mutation, but not in CASQ2-/- / E4872Q+/- hearts. These observations indicate that targeting the luminal calcium sensor of RyR2 represents a new therapeutic strategy for suppressing CPVT and other calcium mediated cardiac arrhythmias. (Support by NIH R01HL75210)