RyR Isoforms Research Articles

Effect of Human RyR2 CPVT Mutations on Interaction with Calmodulin

The ryanodine receptor (RyR) is a member of a family of intracellular calcium release channels that regulate calcium efflux from intracellular stores. The RyR2 isoform is most abundant in the heart and plays a key role in cardiac muscle excitation-contraction coupling. Clusters of mutations associated with the inherited arrhythmogenic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), have been found in specific regions throughout RyR2, a large protein of ∼5000 amino acids. Many of these CPVT mutations (total >100) are thought to occur in significant functional domains and result in the dysregulation of RyR channel function.One such region of RyR2 is believed to comprise a calmodulin (CaM) interaction site and two EF hand motifs. The calcium-sensitive binding of CaM has been shown to regulate the opening of RyR. Hence, examining the RyR2 interaction with CaM and the potential effects of CPVT mutations on this binding may help reveal mutation-dependent mechanisms of channel dysfunction.We have prepared bacterial expression plasmid constructs containing the wild-type human RyR2 CaM-interacting domain and introduced a series of CPVT mutations that have previously been identified to occur within this region. Expression and purification of the corresponding recombinant fusion proteins has enabled calcium-dependent binding of CaM to be determined with all these constructs. Examining the distinct functional role of calcium concentration on CaM binding kinetics and further comparative structural analyses of the wild-type and mutant RyR2 domains may help reveal the specific effect(s) that CaM-mediated regulation may have in mediating CPVT-linked arrhythmogenesis.

Different Capabilities for CICR of Skeletal Muscle of Amphibians and Mammals, Demonstrated through the Response to Artificial Ca Sparks

To probe CICR (Endo, Phys. Rev. 2009), single skeletal muscle cells were stimulated with artificial Ca2+ sparks generated by spot 2-photon breakdown of NDBF-EGTA, while their Ca2+ response was monitored by confocal imaging of fluorescence of fluo 4FF. The cells had their plasmalemma permeabilized. Sparks were applied either inside cells or slightly outside, a method that avoids photodamage and allows measurement of the triggering [Ca2+] at precisely the time when the cellular response is elicited. Frog skeletal muscle fibers usually responded to stimuli > 1 μM with a propagating, all-or-none Ca2+ wave. The moving wavefront peaked at 3-5 μM; the calculated Ca2+ release flux peaked at 50-100 mM/s and the wave velocity was ∼100 μm/s (and slightly faster in the axial direction). In other cases a “frustrated” response was generated, which stopped after propagating for 20-30 μm, usually following progressive reduction in Ca2+ release flux. In striking contrast, the same stimuli failed to elicit a response when applied to cells of the mouse FDB muscle. Variants of the stimulus included “whorls” (2-P radiation in scribbles that covered regions of ∼3 μm diameter), involving much greater amounts of Ca2+ than an artificial spark. Whether placed outside or inside the cells, even at intensities that caused persistent cell damage, whorls elicited neither the fully propagated nor the frustrated response. These results directly demonstrate that frog muscle is capable of CICR but do not prove a physiologic role, as they were obtained on permeabilized cells. In conclusion, mammalian muscle does not have the CICR capability, which other works have associated with the presence of RyR isoform 3 (Pouvreau et al., PNAS 2007; Legrand et al., J. Physiol. 2008). Supported by NIAMS and NHLBI (NIH).

Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation

Ryanodine receptors (RyR) amplify activity-dependent calcium influx via calcium-induced calcium release. Calcium signals trigger postsynaptic pathways in hippocampal neurons that underlie synaptic plasticity, learning, and memory. Recent evidence supports a role of the RyR2 and RyR3 isoforms in these processes. Along with calcium signals, brain-derived neurotrophic factor (BDNF) is a key signaling molecule for hippocampal synaptic plasticity and spatial memory. Upon binding to specific TrkB receptors, BDNF initiates complex signaling pathways that modify synaptic structure and function. Here, we show that BDNF-induced remodeling of hippocampal dendritic spines required functional RyR. Additionally, incubation with BDNF enhanced the expression of RyR2, RyR3, and PKMζ, an atypical protein kinase C isoform with key roles in hippocampal memory consolidation. Consistent with their increased RyR protein content, BDNF-treated neurons generated larger RyR-mediated calcium signals than controls. Selective inhibition of RyR-mediated calcium release with inhibitory ryanodine concentrations prevented the PKMζ, RyR2, and RyR3 protein content enhancement induced by BDNF. Intrahippocampal injection of BDNF or training rats in a spatial memory task enhanced PKMζ, RyR2, RyR3, and BDNF hippocampal protein content, while injection of ryanodine at concentrations that stimulate RyR-mediated calcium release improved spatial memory learning and enhanced memory consolidation. We propose that RyR-generated calcium signals are key features of the complex neuronal plasticity processes induced by BDNF, which include increased expression of RyR2, RyR3, and PKMζ and the spine remodeling required for spatial memory formation.

Out of a Cell into This Darkened Space

Out of a Cell into This Darkened Space

Mapping the Ryanodine Receptor FK506-binding Protein Subunit Using Fluorescence Resonance Energy Transfer

The 12-kDa FK506-binding proteins (FKBP12 and FKBP12.6) are regulatory subunits of ryanodine receptor (RyR) Ca(2+) release channels. To investigate the structural basis of FKBP interactions with the RyR1 and RyR2 isoforms, we used site-directed fluorescent labeling of FKBP12.6, ligand binding measurements, and fluorescence resonance energy transfer (FRET). Single-cysteine substitutions were introduced at five positions distributed over the surface of FKBP12.6. Fluorescent labeling at position 14, 32, 49, or 85 did not affect high affinity binding to the RyR1. By comparison, fluorescent labeling at position 41 reduced the affinity of FKBP12.6 binding by 10-fold. Each of the five fluorescent FKBPs retained the ability to inhibit [(3)H]ryanodine binding to the RyR1, although the maximal extent of inhibition was reduced by half when the label was attached at position 32. The orientation of FKBP12.6 bound to the RyR1 and RyR2 was examined by measuring FRET from the different labeling positions on FKBP12.6 to an acceptor attached within the RyR calmodulin subunit. FRET was dependent on the position of fluorophore attachment on FKBP12.6; however, for any given position, the distance separating donors and acceptors bound to RyR1 versus RyR2 did not differ significantly. Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model.

Chapter 5 - Regulation of Ryanodine Receptor Ion Channels Through Posttranslational Modifications

Publisher Summary The ryanodine receptors (RyRs) are cation-selective channels that release Ca2+ from an intracellular Ca2+ storing compartment, the endo/sarcoplasmic reticulum, during an action potential in a process known as “excitation–contraction (E–C) coupling.” The released Ca2+ ions regulate a wide variety of biological functions. This chapter focuses on the regulation of the skeletal muscle and cardiac muscle RyRs by protein kinases and redox active species. The RyRsare 2200 kDa Ca2+-gated ion channels that are regulated, in addition to Ca2+ and endogenous effectors such as Mg2+ and ATP, by cAMP-dependent protein kinase A, calmodulin-dependent kinase II, protein kinase C, and protein phosphatases 1 and 2A. Thiols that serve as targets for reactive oxygen and nitrogen molecules determine the redox state and modulate the activity of the RyRs. There are three mammalian RyR isoforms. RyR1 is found in the sarcoplasmic reticulum (SR) membrane of skeletal muscle, while RyR2 is present in heart muscle. In mammalian cells, RyR3 is coexpressed with one or two of the other RyR isoforms at low levels. All three isoforms are present in smooth muscle and brain. The third mammalian RyR isoform, RyR3, contains putative phosphorylation and redox active sites; however, coexpression with the other mammalian RyR isoforms has hindered establishment of a specific cellular role.

FRET Detection of Calmodulin Binding and Structural Rearrangements Within the Cardiac RyR2 Calcium Release Channel

Calmodulin (CaM) binds to a conserved domain of the ryanodine receptor isoforms expressed in skeletal muscle (RyR1) and cardiac muscle (RyR2) to evoke isoform-specific changes in channel gating. To better understand CaM's interactions with the RyR2 isoform, we are using fluorescence resonance energy transfer (FRET) to define the orientation and kinetics of CaM binding, and to resolve structural rearrangements linked to channel regulation. A FRET donor was targeted to the RyR2 cytoplasmic assembly by preincubating cardiac sarcoplasmic reticulum membranes with a fluorescent-labeled FKBP12.6 (F-FKBP). An acceptor fluorophore was attached within the N-lobe of CaM (F-CaM). A decrease in F-FKBP fluorescence upon addition of F-CaM provided a specific, real-time readout of CaM binding to the RyR2, despite the presence of additional non-RyR CaM targets in the cardiac membranes. FRET demonstrated that the affinity of F-CaM binding to RyR2 was greater in 100 μM than in 30 nM Ca2+. The maximal FRET observed in the presence of saturating [F-CaM] increased as a function of [Ca2+] (30 nM to 1 mM). The Ca2+ dependence of this increase in FRET was similar to the Ca2+ dependence of [3H]ryanodine binding to RyR2 assayed in equivalent media (KCa ∼5 μM). A marked decrease in FRET between FKBP12.6 and CaM was observed when the acceptor was shifted from CaM's N-lobe to CaM's C-lobe. We conclude that CaM binds to the RyR2 in an extended conformation, with its N-lobe oriented nearest to the FKBP12.6 subunit. CaM's conformation and orientation when bound to the RyR2 are therefore similar to what has been demonstrated previously for the RyR1 isoform (Cornea et al., 2009). Ca2+ dependent changes in FRET between FKBP12.6 and CaM may reflect structural changes within the RyR2 linked to channel activation by Ca2+.

Deviant Ryanodine Receptor-Mediated Calcium Release Resets Synaptic Homeostasis in Presymptomatic 3xTg-AD Mice

Presenilin mutations result in exaggerated endoplasmic reticulum (ER) calcium release in cellular and animal models of Alzheimer's disease (AD). In this study, we examined whether dysregulated ER calcium release in young 3xTg-AD neurons alters synaptic transmission and plasticity mechanisms before the onset of histopathology and cognitive deficits. Using electrophysiological recordings and two-photon calcium imaging in young (6-8 weeks old) 3xTg-AD and non-transgenic (NonTg) hippocampal slices, we show a marked increase in ryanodine receptor (RyR)-evoked calcium release within synapse-dense regions of CA1 pyramidal neurons. In addition, we uncovered a deviant contribution of presynaptic and postsynaptic ryanodine receptor-sensitive calcium stores to synaptic transmission and plasticity in 3xTg-AD mice that is not present in NonTg mice. As a possible underlying mechanism, the RyR2 isoform was found to be selectively increased more than fivefold in the hippocampus of 3xTg-AD mice relative to the NonTg controls. These novel findings demonstrate that 3xTg-AD CA1 neurons at presymptomatic ages operate under an aberrant, yet seemingly functional, calcium signaling and synaptic transmission system long before AD histopathology onset. These early signaling alterations may underlie the later synaptic breakdown and cognitive deficits characteristic of later stage AD.

A Computational Analysis of Localized Ca2+-Dynamics Generated by Heterogeneous Release Sites

We investigate the role of heterogeneous expression of IP3R and RyR in generating diverse elementary Ca2+ signals. It has been shown empirically (Wojcikiewicz and Luo in Mol. Pharmacol. 53(4):656-662, 1998; Newton et al. in J. Biol. Chem. 269(46):28613-28619, 1994; Smedt et al. in Biochem. J. 322(Pt. 2):575-583, 1997) that tissues express various proportions of IP3 and RyR isoforms and this expression is dynamically regulated (Parrington et al. in Dev. Biol. 203(2):451-461, 1998; Fissore et al. in Biol. Reprod. 60(1):49-57, 1999; Tovey et al. in J. Cell Sci. 114(Pt. 22):3979-3989, 2001). Although many previous theoretical studies have investigated the dynamics of localized calcium release sites (Swillens et al. in Proc. Natl. Acad. Sci. U.S.A. 96(24):13750-13755, 1999; Shuai and Jung in Proc. Natl. Acad. Sci. U.S.A. 100(2):506-510, 2003a; Shuai and Jung in Phys. Rev. E, Stat. Nonlinear Soft Matter Phys. 67(3 Pt. 1):031905, 2003b; Thul and Falcke in Biophys. J. 86(5):2660-2673, 2004; DeRemigio and Smith in Cell Calcium 38(2):73-86, 2005; Nguyen et al. in Bull. Math. Biol. 67(3):393-432, 2005), so far all such studies focused on release sites consisting of identical channel types. We have extended an existing mathematical model (Nguyen et al. in Bull. Math. Biol. 67(3):393-432, 2005) to release sites with two (or more) receptor types, each with its distinct channel kinetics. Mathematically, the release site is represented by a transition probability matrix for a collection of nonidentical stochastically gating channels coupled through a shared Ca2+ domain. We demonstrate that under certain conditions a previously defined mean-field approximation of the coupling strength does not accurately reproduce the release site dynamics. We develop a novel approximation and establish that its performance in these instances is superior. We use this mathematical framework to study the effect of heterogeneity in the Ca2+-regulation of two colocalized channel types on the release site dynamics. We consider release sites consisting of channels with both Ca2+-activation and inactivation ("four-state channels") and channels with Ca2+-activation only ("two-state channels") and show that for the appropriate parameter values, synchronous channel openings within a release site with any proportion of two-state to four-state channels are possible, however, the larger the proportion of two-state channels, the more sensitive the dynamics are to the exact spatial positioning of the channels and the distance between channels. Specifically, the clustering of even a small number of two-state channels interferes with puff/spark termination and increases puff durations or leads to a tonic response.

FRET-based mapping of calmodulin bound to the RyR1 Ca 2+ release channel

Calmodulin (CaM) functions as a regulatory subunit of ryanodine receptor (RyR) channels, modulating channel activity in response to changing [Ca(2+)](i). To investigate the structural basis of CaM regulation of the RyR1 isoform, we used site-directed labeling of channel regulatory subunits and fluorescence resonance energy transfer (FRET). Donor fluorophore was targeted to the RyR1 cytoplasmic assembly by preincubating sarcoplasmic reticulum membranes with a fluorescent FK506-binding protein (FKBP), and FRET was determined following incubations in the presence of fluorescent CaMs in which acceptor fluorophore was attached within the N lobe, central linker, or C lobe. Results demonstrated strong FRET to acceptors attached within CaM's N lobe, whereas substantially weaker FRET was observed when acceptor was attached within CaM's central linker or C lobe. Surprisingly, Ca(2+) evoked little change in FRET to any of the 3 CaM domains. Donor-acceptor distances derived from our FRET measurements provide insights into CaM's location and orientation within the RyR1 3D architecture and the conformational switching that underlies CaM regulation of the channel. These results establish a powerful new approach to resolving the structure and function of RyR channels.

Ryanodine Receptor Structure: Progress and Challenges

Ryanodine Receptor Structure: Progress and Challenges

A new role for type 1 ryanodine receptor

Type 1 ryanodine receptors (RyR1) are the second most common isoform found in neurons. We hypothesize that in nerve terminals from neurohypophysis, L-type Ca2+ channels are coupled to RyR1 in the same way found in EC coupling in skeletal muscle. In these nerve terminals we showed (J.Neuroscience 2006, 26 -7565) that L-type channels are the sensors of membrane potential for Voltage Induced Ca2+ Release (VICaR), independently of their role as Ca2+ current carriers and that RyRs are the effectors through which Ca2+ is released into the cytosol. Here we wished to determine which RyR isoforms are responsible for VICaR. We studied Ca2+ syntillas (scintilla, L., spark in synaptic structure, a nerve terminal) in two different mutant mice with a knock-in mutation in RyR1 in physiological extracellular [Ca2+]. In the first, R163C, which has a gain of function phenotype, described as leaky and causes Central Core Disease and Malignant Hyperthermia in humans, depolarization to -60mV from -80mV caused a global increase in [Ca2+]. This increase was not seen in WT where such depolarization caused only an increase in syntilla frequency. In the second RyR1 mutant, I4898T, which has a loss of function phenotype, described as EC uncoupling, and causes Central Core Disease in humans, there was not only an absence of the global increase in [Ca2+] but also a significant decrease in syntilla frequency at −60mV compared to −80mV. Moreover basal [Ca2+] in the terminals, measured using fura-2, was significantly lower in I4898T. Finally, depolarization to 0mV resulted in a 4 fold decrease in the Ca2+ transient compared to WT. These data shows, for the first time in a preparation other than skeletal muscle, that RyR1 is involved in determining global [Ca2+] both at rest and by a process of VICaR, upon depolarization.

A FRET-based Assay To Measure Molecular Distances Within The Ryanodine Receptor Type 1 (RyR1)

The type 1 ryanodine receptor (RyR1) is an intracellular Ca2+-release channel that mediates excitation contraction coupling in skeletal muscle. This enormous homotetrameric protein has a subunit molecular weight of 565 kDa and is associated with numerous regulatory proteins that modulate its function in vivo. Understanding the structure and conformational dynamics of this immense macromolecular complex is a key topic in skeletal muscle biology. In this report, a novel structural assay has been devised that relies upon Forster resonance energy transfer (FRET) to measure distances between defined primary sequence elements of RyR1. These FRET measurements require site-specific incorporation of a fluorescence donor and a fluorescence acceptor into the primary sequence of RyR1. In this system, green fluorescent protein (GFP) fused to the N-terminus of RyR1 acts as the fluorescence donor. The FRET acceptor is an affinity reagent that site-specifically binds to poly-histidine segments (i.e. His tags) engineered into RyR1 where it can accept fluorescence energy from the N-terminal GFP. This report describes the characterization of the FRET acceptor as well as the recombinant His-tagged GFP-RyR1 fusion proteins used for these measurements. In addition, experiments measuring FRET from the N-terminal GFP to the FRET acceptor targeted to His tags introduced into 3 primary sequence elements poorly conserved among the 3 RyR isoforms (i.e. divergent regions) are described. (Supported by NIH grants K01ARO52120 and R21ARO56406).

Structural Characterization of FKBP Interactions with RyR Channels Using Site-Directed Fluorescent Labeling and FRET

The small FK506-binding proteins (FKBP12/12.6) tightly bind to ryanodine receptor (RyR) channels and may stabilize channels in a closed conformation. To investigate the structural basis of FKBP12.6 binding to the RyR1 and RyR2 isoforms, we used cysteine mutagenesis, fluorescent labeling, direct-binding measurements, and FRET. Single cysteines were introduced at five positions distributed over the surface of FKBP12.6 (T14C, N32C, D41C, R49C, and T85C). Results showed that each of the five FKBP12.6 mutants retained high-affinity binding to the RyR1. Furthermore, high-affinity binding was retained following the covalent attachment of a 720 Da fluorescent donor at four of the five positions (14, 32, 49, and 85), suggesting that these positions are removed from the major RyR1 binding interface. By comparison, attachment of the fluorescent donor at one position (41) resulted in a marked decrease in FKBP12.6 binding. FRET from the different donor-labeled FKBPs to an acceptor attached within the RyR CaM subunit was examined to determine the orientation of FKBP12.6 bound to the RyR1 and RyR2. Results showed that FRET was dependent on the donor's position on FKBP12.6, and that the rank order of FRET efficiency from the different positions was the same for the two RyR isoforms (position 49 > 85 ≥ 14 > 32). These results indicate that in binding either RyR1 or RyR2, FKBP12.6 is oriented such that position 49 is nearest and 32 furthest from the RyR CaM binding site. Together, our results are consistent with the model of FKBP binding proposed by Samso and coworkers (2006), and point to loop 39–46 as a key component of the RyR binding interface.

Comprehensive behavioral phenotyping of ryanodine receptor type3 (RyR3) knockout mice: Decreased social contact duration in two social interaction tests

Dynamic regulation of the intracellular Ca2+ concentration is crucial for various neuronal functions such as synaptic transmission and plasticity, and gene expression. Ryanodine receptors (RyRs) are a family of intracellular calcium release channels that mediate calcium-induced calcium release from the endoplasmic reticulum. Among the three RyR isoforms, RyR3 is preferentially expressed in the brain especially in the hippocampus and striatum. To investigate the behavioral effects of RyR3 deficiency, we subjected RyR3 knockout (RyR3–/–) mice to a battery of behavioral tests. RyR3–/– mice exhibited significantly decreased social contact duration in two different social interaction tests, where two mice can freely move and make contacts with each other. They also exhibited hyperactivity and mildly impaired prepulse inhibition and latent inhibition while they did not show significant abnormalities in motor function and working and reference memory tests. These results indicate that RyR3 has an important role in locomotor activity and social behavior.

Role of Ryanodine Receptor Subtypes in Initiation and Formation of Calcium Sparks in Arterial Smooth Muscle:Comparisonwith Striated Muscle

Calcium sparks represent local, rapid, and transient calcium release events from a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum. In arterial smooth muscle cells (SMCs), calcium sparks activate calcium-dependent potassium channels causing decrease in the global intracellular [Ca2+] and oppose vasoconstriction. This is in contrast to cardiac and skeletal muscle, where spatial and temporal summation of calcium sparks leads to global increases in intracellular [Ca2+] and myocyte contraction. We summarize the present data on local RyR calcium signaling in arterial SMCs in comparison to striated muscle and muscle-specific differences in coupling between L-type calcium channels and RyRs. Accordingly, arterial SMC Cav1.2 L-type channels regulate intracellular calcium stores content, which in turn modulates calcium efflux though RyRs. Downregulation of RyR2 up to a certain degree is compensated by increased SR calcium content to normalize calcium sparks. This indirect coupling between Cav1.2 and RyR in arterial SMCs is opposite to striated muscle, where triggering of calcium sparks is controlled by rapid and direct cross-talk between Cav1.1/Cav1.2 L-type channels and RyRs. We discuss the role of RyR isoforms in initiation and formation of calcium sparks in SMCs and their possible molecular binding partners and regulators, which differ compared to striated muscle.

MCF-7 breast carcinoma cells express ryanodine receptor type 1: functional characterization and subcellular localization

Breast carcinoma-derived MCF-7 cells are frequently used in biomedical research. However, few reports exist regarding the characterization of signaling mechanisms in these cancerous cells involved in intracellular Ca(2+) dynamics. Consequently, the aim of these experiments was to characterize the ryanodine receptor/Ca(2+) release channel (RyR) present in MCF-7 cells. Ryanodine (100 nM), cADPR (5 microM), and caffeine (10 mM) promoted cytoplasmic Ca(2+) mobilization; in contrast, ryanodine at inhibitory concentration (100 microM) decreased the basal Ca(2+) level. Fluorescent probes demonstrated that RyR is located mainly in endomembranes. Some degree of co-localization with inositol trisphosphate receptor (IP(3)R) was observed, whereas coincidence with thapsigargin-sensitive Ca(2+)-ATPase (SERCA) was more limited. Molecular cloning resulted in the detection exclusively of RyR isoform 1. For the first time, it is shown that MCF-7 cells express functional RyR.

Ryanodine Receptor Activation by Cav1.2 Is Involved in Dendritic Cell Major Histocompatibility Complex Class II Surface Expression

Dendritic cells express the skeletal muscle ryanodine receptor (RyR1), yet little is known concerning its physiological role and activation mechanism. Here we show that dendritic cells also express the Ca(v)1.2 subunit of the L-type Ca(2+) channel and that release of intracellular Ca(2+) via RyR1 depends on the presence of extracellular Ca(2+) and is sensitive to ryanodine and nifedipine. Interestingly, RyR1 activation causes a very rapid increase in expression of major histocompatibility complex II molecules on the surface of dendritic cells, an effect that is also observed upon incubation of mouse BM12 dendritic cells with transgenic T cells whose T cell receptor is specific for the I-A(bm12) protein. Based on the present results, we suggest that activation of the RyR1 signaling cascade may be important in the early stages of infection, providing the immune system with a rapid mechanism to initiate an early response, facilitating the presentation of antigens to T cells by dendritic cells before their full maturation.

Abstract 338: Cardiac-type Calcium Release Mechanism in Cardiac Precursor Cells

Cardiac progenitor cells (CPCs) form three-dimensional structure named cardiospheres (CSs). While CSs are seen as partially committed cardiac precursors, it is unknown whether they already display cardiac-type molecular functions. Whereas IP3-mediated Ca 2+ release is a shared property of many cell types, Ca + release through RyR channels is muscle specific. Aims: To test if cardiac-type Ca 2+ release mechanism is present in CPCs and whether it may develop during the CSs stage. Methods: Cells arose from murine cardiac explants in culture were studied prior to CSs formation (pre-CSs) and after expansion of CSs (post-CSs). Ca 2+ transients were detected in wide optical confocal fields by Fluo4-AM fluorescence. RyR- and IP3-R-mediated Ca 2+ release from intracellular stores was tested by exposures to caffeine (CAF, 10 mM) or ATP (200 μM) respectively in Ca 2+ -free conditions. The expression of the cardiac-specific RyR isoform (RyR2) was tested by immunolabeling and western-blot analysis. Results: In isolated cells CAF-induced response was almost exclusive of post-CSs cells (22.4 % vs. 3.62 %; p<0.05); ATP-induced response was already present in pre-CSs cells and showed only a small increase in post-CSs ones (94.3 % vs 86.1 % p<0.05). The ratio between post-CSs and pre-CSs responses was 6.2 for CAF and 1.1 for ATP. Both CAF and ATP responses were suppressed by the SERCA inhibitor CPA (50 μM), thus confirming intracellular stores as the Ca 2+ source. ATP response only was suppressed by the IP3-R blocker 2APB (10 μM). Ryanodine (20 μM) prevented the response to CAF, but not to ATP. In post-CSs RyR2 protein levels were higher than in pre-CSs and similar to those of adult myocytes. Immunoistochemistry analysis of post-CSs cells results in a distribution of RyR2 expression consistent with immature neonatal cardiomyocytes previously described. Conclusions: At variance with IP3-mediated signaling, RyR mediated Ca 2+ release develops during maturation within the CSs environment, along with expression of the cardiac RyR isoform. Detection of caffeine-induced Ca 2+ responses may be useful in identifying cardio-specific functional maturation in progenitor cell populations.

Ischemia enhances activation by Ca2+ and redox modification of ryanodine receptor channels from rat brain cortex.

Cerebral ischemia stimulates Ca2+ influx and thus increases neuronal intracellular free [Ca2+]. Using a rat model of cerebral ischemia without recirculation, we tested whether ischemia enhances the activation by Ca2+ of ryanodine receptor (RyR) channels, a requisite feature of RyR-mediated Ca2+-induced Ca2+ release (CICR). To this aim, we evaluated how single RyR channels from endoplasmic reticulum vesicles, fused into planar lipid bilayers, responded to cytoplasmic [Ca2+] changes. Endoplasmic reticulum vesicles were isolated from the cortex of rat brains incubated without blood flow for 5 min at 37 degrees C (ischemic) or at 4 degrees C (control). Ischemic brains displayed increased oxidative intracellular conditions, as evidenced by a lower ratio (approximately 130:1) of reduced/oxidized glutathione than controls (approximately 200:1). Single RyR channels from ischemic or control brains displayed the same three responses to Ca2+ reported previously, characterized by low, moderate, or high maximal activity. Relative to controls, RyR channels from ischemic brains displayed with increased frequency the high activity response and with lower frequency the low activity response. Both control and ischemic cortical vesicles contained the RyR2 and RyR3 isoforms in a 3:1 proportion, with undetectable amounts of RyR1. Ischemia reduced [3H]ryanodine binding and total RyR protein content by 35%, and increased at least twofold endogenous RyR2 S-nitrosylation and S-glutathionylation without affecting the corresponding RyR3 endogenous levels. In vitro RyR S-glutathionylation but not S-nitrosylation favored the emergence of high activity channels. We propose that ischemia, by enhancing RyR2 S-glutathionylation, allows RyR2 to sustain CICR; the resulting amplification of Ca2+ entry signals may contribute to cortical neuronal death.