Ca(2+)-induced Ca2+ release in myocytes from dyspedic mice lacking the type-1 ryanodine receptor.
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PROVIDER: S-EPMC394359 | BioStudies | 1995-01-01T00:00:00Z
REPOSITORIES: biostudies
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Project description:The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.
1998-01-01 | S-EPMC1170669 | BioStudies
Project description:KEY POINTS:Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non-dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z-axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. ABSTRACT:Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z-axis and obscured detection by 2D techniques. Non-overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single 'rogue' RyR. Internal CRUs were more tightly packed along z-lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ?75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t-tubules, which enabled quantification of dyadic and non-dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.
| S-EPMC6332759 | BioStudies
Project description:Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca2+] and intra-SR [Ca2+]SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10?M) increased Ca2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10?M) abolished the cAMP-dependent increase of SR Ca2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca2+ leak.
| S-EPMC5337148 | BioStudies
Project description:Calexcitin (CE), a Ca2+- and GTP-binding protein, which is phosphorylated during memory consolidation, is shown here to co-purify with ryanodine receptors (RyRs) and bind to RyRs in a calcium-dependent manner. Nanomolar concentrations of CE released up to 46% of the 45Ca label from microsomes preloaded with 45CaCl2. This release was Ca2+-dependent and was blocked by antibodies against the RyR or CE, by the RyR inhibitor dantrolene, and by a seven-amino-acid peptide fragment corresponding to positions 4689-4697 of the RyR, but not by heparin, an Ins(1,4,5)P3-receptor antagonist. Anti-CE antibodies, in the absence of added CE, also blocked Ca2+ release elicited by ryanodine, suggesting that the CE and ryanodine binding sites were in relative proximity. Calcium imaging with bis-fura-2 after loading CE into hippocampal CA1 pyramidal cells in hippocampal slices revealed slow, local calcium transients independent of membrane depolarization. Calexcitin also released Ca2+ from liposomes into which purified RyR had been incorporated, indicating that CE binding can be a proximate cause of Ca2+ release. These results indicated that CE bound to RyRs and suggest that CE may be an endogenous modulator of the neuronal RyR.
1999-01-01 | S-EPMC1220376 | BioStudies
Project description:The induction of both long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission entails pre- and postsynaptic Ca2+ signals, which represent transient increments in cytoplasmic free Ca2+ concentration. In diverse synapse types, Ca2+ release from intracellular stores contributes to amplify the Ca2+ signals initially generated by activation of neuronal Ca2+ entry pathways. Here, we used hippocampal slices from young male rats to evaluate whether pharmacological activation or inhibition of Ca2+ release from the endoplasmic reticulum (ER) mediated by ryanodine receptor (RyR) channels modifies LTD induction at Schaffer collateral-CA1 synapses. Pre-incubation of slices with ryanodine (1 ?M, 1 h) or caffeine (1 mM, 30 min) to promote RyR-mediated Ca2+ release facilitated LTD induction by low frequency stimulation (LFS), but did not affect the amplitude of synaptic transmission, the profiles of field excitatory postsynaptic potentials (fEPSP) or the paired-pulse (PP) responses. Conversely, treatment with inhibitory ryanodine (20 ?M, 1 h) to suppress RyR-mediated Ca2+ release prevented LTD induction, but did not affect baseline synaptic transmission or PP responses. Previous literature reports indicate that LTD induction requires presynaptic CaMKII activity. We found that 1 h after applying the LTD induction protocol, slices displayed a significant increase in CaMKII phosphorylation relative to the levels exhibited by un-stimulated (naïve) slices. In addition, LTD induction (1 h) enhanced the phosphorylation of the presynaptic protein Synapsin I at a CaMKII-dependent phosphorylation site, indicating that LTD induction stimulates presynaptic CaMKII activity. Pre-incubation of slices with 20 ?M ryanodine abolished the increased CaMKII and Synapsin I phosphorylation induced by LTD, whereas naïve slices pre-incubated with inhibitory ryanodine displayed similar CaMKII and Synapsin I phosphorylation levels as naïve control slices. We posit that inhibitory ryanodine suppressed LTD-induced presynaptic CaMKII activity, as evidenced by the suppression of Synapsin I phosphorylation induced by LTD. Accordingly, we propose that presynaptic RyR-mediated Ca2+ signals contribute to LTD induction at Schaffer collateral-CA1 synapses.
| S-EPMC6232521 | BioStudies
Project description:Aims:In ventricular myocytes from humans and large mammals, the transverse and axial tubular system (TATS) network is less extensive than in rodents with consequently a greater proportion of ryanodine receptors (RyRs) not coupled to this membrane system. TATS remodelling in heart failure (HF) and after myocardial infarction (MI) increases the fraction of non-coupled RyRs. Here we investigate whether this remodelling alters the activity of coupled and non-coupled RyR sub-populations through changes in local signalling. We study myocytes from patients with end-stage HF, compared with non-failing (non-HF), and myocytes from pigs with MI and reduced left ventricular (LV) function, compared with sham intervention (SHAM). Methods and results:Single LV myocytes for functional studies were isolated according to standard protocols. Immunofluorescent staining visualized organization of TATS and RyRs. Ca2+ was measured by confocal imaging (fluo-4 as indicator) and using whole-cell patch-clamp (37°C). Spontaneous Ca2+ release events, Ca2+ sparks, as a readout for RyR activity were recorded during a 15 s period following conditioning stimulation at 2 Hz. Sparks were assigned to cell regions categorized as coupled or non-coupled sites according to a previously developed method. Human HF myocytes had more non-coupled sites and these had more spontaneous activity than in non-HF. Hyperactivity of these non-coupled RyRs was reduced by Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition. Myocytes from MI pigs had similar changes compared with SHAM controls as seen in human HF myocytes. As well as by CaMKII inhibition, in MI, the increased activity of non-coupled sites was inhibited by mitochondrial reactive oxygen species (mito-ROS) scavenging. Under adrenergic stimulation, Ca2+ waves were more frequent and originated at non-coupled sites, generating larger Na+/Ca2+ exchange currents in MI than in SHAM. Inhibition of CaMKII or mito-ROS scavenging reduced spontaneous Ca2+ waves, and improved excitation-contraction coupling. Conclusions:In HF and after MI, RyR microdomain re-organization enhances spontaneous Ca2+ release at non-coupled sites in a manner dependent on CaMKII activation and mito-ROS production. This specific modulation generates a substrate for arrhythmia that appears to be responsive to selective pharmacologic modulation.
| S-EPMC6106102 | BioStudies
Project description:A key feature of signalling in dendritic spines is the synapse-specific transduction of short electrical signals into biochemical responses. Ca2+ is a major upstream effector in this transduction cascade, serving both as a depolarising electrical charge carrier at the membrane and an intracellular second messenger. Upon action potential firing, the majority of spines are subject to global back-propagating action potential (bAP) Ca2+ transients. These transients translate neuronal suprathreshold activation into intracellular biochemical events. Using a combination of electrophysiology, two-photon Ca2+ imaging, and modelling, we demonstrate that bAPs are electrochemically coupled to Ca2+ release from intracellular stores via ryanodine receptors (RyRs). We describe a new function mediated by spine RyRs: the activity-dependent long-term enhancement of the bAP-Ca2+ transient. Spines regulate bAP Ca2+ influx independent of each other, as bAP-Ca2+ transient enhancement is compartmentalized and independent of the dendritic Ca2+ transient. Furthermore, this functional state change depends exclusively on bAPs travelling antidromically into dendrites and spines. Induction, but not expression, of bAP-Ca2+ transient enhancement is a spine-specific function of the RyR. We demonstrate that RyRs can form specific Ca2+ signalling nanodomains within single spines. Functionally, RyR mediated Ca2+ release in these nanodomains induces a new form of Ca2+ transient plasticity that constitutes a spine specific storage mechanism of neuronal suprathreshold activity patterns.
| S-EPMC4476683 | BioStudies
Project description:Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 +/- 8, 334 +/- 15, and 441 +/- 27 pS in symmetric 300 mM Cs(+) were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to <or=100 pS when Cs(+) was replaced with Ca(2+). The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NP(o))-voltage curve was bell shaped, with NP(o) drastically decreasing when voltage was switched from -30 to -40 mV. NP(o) increased when intracellular calcium (Ca(2+)(i)) was raised within 0.1-100 microM to abruptly diminish with higher Ca(2+)(i). Thus maximal activity occurred within the Ca(2+)(i) range found in rat cerebral artery myocytes under physiological conditions. NP(o) was reduced by ruthenium red (80 muM), increased monotonically by caffeine (0.1-5 mM) or ryanodine (0.05-5 microM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 muM), resistant to dantrolene (100 muM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.
| S-EPMC2928634 | BioStudies
Project description:Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and alpha-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and deconvolution. The RyR label formed discrete puncta representing clusters of RyRs or "couplons" around the edges of the myofilaments with a nearest-neighbor spacing of 0.66 +/- 0.06 microm in rat and 0.78 +/- 0.07 microm in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of approximately 0.6 microm(2) in rat and approximately 0.8 microm(2) in human. Although the couplons were in reasonable registration with z-lines, there were discontinuities in the longitudinal position of sarcomeres so that dislocations in the order of RyR clusters occurred. There was approximately 53% longitudinal registration of RyR clusters, suggesting a nonrandom placement of couplons around the sarcomere. These data can explain the spherical propagation of Ca(2+) waves and provide quantitative 3D data sets needed for accurate modeling of cardiac Ca(2+)-induced Ca(2+) release. By quantifying labeling intensity in rat ventricular myocytes, a lower limit of 78 RyRs per cluster (on average) was obtained. By modeling the couplon as a disk wrapping around a t-tubule and fitting cluster images, 95% of couplons contained between 120 and 260 RyRs (assuming that RyRs are tight packed with a spacing of 29 nm). Assuming similar labeling efficiency in human, from the fluorescence intensity alone we estimate that human ventricular myocytes contain approximately 30% fewer RyRs per couplon than rat.
| S-EPMC1986595 | BioStudies
Project description:Large-conductance Ca2+ release channels known as ryanodine receptors (RyRs) mediate the release of Ca2+ from an intracellular membrane compartment, the endo/sarcoplasmic reticulum. There are three mammalian RyR isoforms: RyR1 is present in skeletal muscle; RyR2 is in heart muscle; and RyR3 is expressed at low levels in many tissues including brain, smooth muscle, and slow-twitch skeletal muscle. RyRs form large protein complexes comprising four 560-kD RyR subunits, four ?12-kD FK506-binding proteins, and various accessory proteins including calmodulin, protein kinases, and protein phosphatases. RyRs share ?70% sequence identity, with the greatest sequence similarity in the C-terminal region that forms the transmembrane, ion-conducting domain comprising ?500 amino acids. The remaining ?4,500 amino acids form the large regulatory cytoplasmic "foot" structure. Experimental evidence for Ca2+, ATP, phosphorylation, and redox-sensitive sites in the cytoplasmic structure have been described. Exogenous effectors include the two Ca2+ releasing agents caffeine and ryanodine. Recent work describing the near atomic structures of mammalian skeletal and cardiac muscle RyRs provides a structural basis for the regulation of the RyRs by their multiple effectors.
| S-EPMC5715910 | BioStudies