Endoplasmic Reticulum Dysfunction and Ca<Superscript>2<Emphasis Type="Bold">+</Emphasis></Superscript> Deregulation in Isolated CA1 Neurons during Oxygen and Glucose Deprivation

Intracellular calcium ([Ca²⁺]_i) plays a pivotal role in neuronal ischemia. The aim of the present study was to investigate the routes of Ca²⁺ entry during non-excitotoxic oxygen and glucose deprivation (OGD) in acutely dissociated rat CA1 neurons. During OGD the fluo-3/fura red ratio reflecting [Ca²⁺]_i increased rapidly and irreversibly. [Ca²⁺]_i increased to the same degree in Ca²⁺ depleted medium, and also when both the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate (IP₃) receptors were blocked. When the endoplasmic reticulum (ER) Ca²⁺ stores were emptied with thapsigargin no increase in [Ca²⁺]_i was observed independent of extracellular Ca²⁺. The OGD induced Ca²⁺ deregulation in isolated CA1 neurons is not prevented by removing Ca²⁺, or by blocking the IP₃– or RyR receptors. However, when SERCA was blocked, no increase in [Ca²⁺]_i was observed suggesting that SERCA dysfunction represents an important mechanism for ischemic Ca²⁺ overload.     

Suppression of Rad leads to arrhythmogenesis via PKA-mediated phosphorylation of ryanodine receptor activity in the heart

Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca²⁺ channel (LTCC). The aim was to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-Specific dominant-negative suppression of Rad. Transgenic (TG) mice overexpressing dominant-negative mutant Rad (S105N Rad TG) were generated. To measure intracellular Ca²⁺ concentration ([Ca²⁺]_i), we recorded [Ca²⁺]_i transients and Ca²⁺ sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca²⁺]_i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca²⁺ sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca²⁺ content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca²⁺]_i transient. In contrast, phosphorylation of Ser²⁸⁰⁹ on the cardiac ryanodine receptor (RyR2) was significantly enhanced in TG mouse hearts compared with controls. Additionally, the Rad-mediated RyR2 phosphorylation was regulated via a direct interaction of Rad with protein kinase A (PKA).     

Excitation-contraction coupling in heart muscle

We have investigated the links between electrical excitation and contraction in mammalian heart muscle. Using isolated single cells from adult rat ventricle, a whole-cell voltage-clamp technique and quantitative fluorescence microscopy, we have measured simultaneously calcium current (I_ca) and [Ca²⁺]_i (with fura-2). We find that the voltage-dependence of Ica and the [Ca ²⁺]_i-transient and the dependence of [Ca²⁺]_i-transient on depolarization-duration cannot both be readily explained by a simple calcium-induced Ca-release (CICR) mechanism. Additionally, we find that when [Ca²⁺]_i and [Na⁺]_i are at their diastolic levels, activation of the Na-Ca exchange mechanism by depolarization does not measurably trigger the release of Ca²⁺i. Finally, measuring I_ca in adult and neonatal rat heart cells and using the alkaloid ryanodine, we have carried out complementary experiments. These experiments show that there may be an action of ryanodine on I_ca that is independent of [Ca²⁺]_i and independent of a direct action of the alkaloid on the calcium channel itself. Along with experiments of others showing that ryanodine binds to the sarcoplasmic reticulum calcium-release channel/spanning protein complex, our data suggests a model to explain our findings. The model links the calcium channels responsible for Ica to the sarcoplasmic reticulum by means of one or more of the spanning protein(s). Information from the calcium channel can be communitated to the sarcoplasmic reticulum by this route and, presumably, information can move in the opposite direction from the sarcoplasmic reticulum to the calcium channel.     

Ca<Superscript>2+</Superscript> regulation in the absence of the <Emphasis Type="Italic">iplA</Emphasis> gene product in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). The [Ca²⁺]_i-change is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-entry. The significance of the [Ca²⁺]_i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca²⁺-buffers in the cytosol indicates that a [Ca²⁺]_i-increase is required for chemotaxis. Yet, the iplA ^- mutant disrupted in a gene bearing similarity to IP₃-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca²⁺]_i-transient which favours the view that [Ca²⁺]_i-changes are insignificant for chemotaxis.     

Chronic fluoxetine administration increases expression of the L-channel gene Cav1.2 in astrocytes from the brain of treated mice and in culture and augments K-induced increase in [Ca]i

We have recently shown that freshly isolated astrocytes from the mouse brain express mRNA for the L-channel gene Ca_v1.3 to at least the same degree (per mg mRNA) as corresponding neurons. The amount of extracellular Ca²⁺ actually entering cultured astrocytes by its opening is modest, but due to secondary Ca²⁺-mediated stimulation of the ryanodine receptor (RyR) the increase in free cytosolic Ca²⁺ [Ca²⁺]_i is substantial. The other Ca_v1 subtype expressed in brain is Ca_v1.2, which is even expressed in higher density. Although the different primers used for the two genes preclude exact quantitative comparison, the present study suggests that this is also the case in the freshly isolated astrocytes and neurons, which express equal Ca_v1.2 densities. Again, most of the increase in [Ca²⁺]_i occurred by RyR activity. In contrast to Ca_v1.3 the expression of Ca_v1.2 was greatly increased (doubled) after two weeks of treatment with fluoxetine hydrochloride (10 mg/kg). Accordingly [Ca²⁺]_i in cultured astrocytes exposed to the addition of 10–60 mM KCl increased substantially in cultured astrocytes treated chronically with fluoxetine with the lag time until the effect was observed depending upon the fluoxetine concentration. This effect was inhibited by nifedipine or siRNA against Ca_v1.2. The increase in K⁺-induced rise in [Ca²⁺]_i after fluoxetine treatment is directly opposite to a decrease in [Ca²⁺]_i after treatment with any of the anti-bipolar drugs lithium, carbamazepine or valproic acid, due to reduced capacitative Ca²⁺ influx. We have previously shown a similar effect after fluoxetine treatment, but it becomes overridden by the Ca_v1.2 up-regulation.     

?-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca²⁺ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca²⁺ transients and reduced Ca²⁺ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca²⁺ and Zn²⁺ ([Ca²⁺]_i and [Zn²⁺]_i), and spatio-temporal properties of the Ca²⁺ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca²⁺]_i-handling regulators, such as Na⁺/Ca²⁺ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca²⁺]_i and [Zn²⁺]_i, increased intracellular Zn²⁺ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca²⁺ handling regulators, and prevention of Ca²⁺ leak, and thereby normalization of both [Ca²⁺]_i and [Zn²⁺]_i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.     

Controlling the urge for a Ca2+ surge: all-or-none Ca2+ release in neurons

Changes in the intracellular calcium concentration ([Ca²⁺]_i) convey signals that are essential to the life and death of neurons. Ca²⁺-induced Ca²⁺-release (CICR), a process in which a modest elevation in [Ca²⁺]_i is amplified by a secondary release of Ca²⁺ from stores within the cell, plays a prominent role in shaping neuronal [Ca²⁺]_i signals. When CICR becomes regenerative, an explosive increase in [Ca²⁺]_i generates a Ca²⁺ wave that spreads throughout the cell. A discrete threshold controls activation of this all-or-none behavior and cellular context adjusts the threshold. Thus, the store acts as a switch that determines whether a given pattern of electrical activity will produce a local or global Ca²⁺ signal. This gatekeeper function seems to control some forms of Ca²⁺-triggered plasticity in neurons. BioEssays 21:743–750, 1999. © 1999 John Wiley & Sons, Inc.     

Examination of the Effects of Heterogeneous Organization of RyR Clusters,Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, [Ca²⁺]_i, regulate the contractile function of cardiac muscle cells. Measuring [Ca²⁺]_i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca²⁺]_i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca²⁺ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca²⁺]_i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca²⁺]_i transient and of the simulated fluorescence intensity signal, F/F₀, reached values similar to that found in the literature ([Ca²⁺]_i ≈1 μM; F/F₀≈5.5). However, our model predicted the variation in [Ca²⁺]_i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F₀ signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca²⁺]_i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca²⁺]_i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca²⁺]_i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.     

Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca²⁺ microelectrodes in myotubes expressing wild-type RyR1 (_WTRyR1) or RyR1 with one of four common MH mutations (_MHRyR1), we measured resting intracellular Ca²⁺ concentration ([Ca²⁺]_i). Changes in resting [Ca²⁺]_i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the _MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca²⁺]_i than those expressing _WTRyR1. Exposure of myotubes expressing _MHRyR1s to ryanodine (500 µM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 µM) had no effects on their resting [Ca²⁺]_i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca²⁺]_i was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca²⁺]_i in myotubes expressing _MHRyR1s was significantly greater than that for _WTRyR1. From these data, we propose that the high resting myoplasmic [Ca²⁺]_i in _MHRyR1 expressing myotubes is due in part to a related structural conformation of _MHRyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum. ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum     

Neuron-derived factors negatively modulate ryanodine receptor-mediated calcium release in cultured mouse astrocytes

Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) produced by ryanodine receptor (RyR) agonist, caffeine (caf), and ionotropic agonists: N-methyl-d-aspartate (NMDA) receptor (NMDAR) agonist, NMDA and P2X7 receptor (P2X7R) agonist, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP) were measured in cultured mouse cortical astrocytes loaded with the fluorescent calcium indicator Fluo3-AM in a confocal laser scanning microscope. In mouse astrocytes cultured in standard medium (SM), treatment with caf increased [Ca²⁺]_i, with a peak response occurring about 10 min after stimulus application. Peak responses to NMDA or BzATP were observed about <1 min and 4.5 min post stimulus, respectively. Co-treatment with NMDA or BzATP did not alter the peak response to caf in astrocytes cultured in SM, the absence of the effects being most likely due to asynchrony between the response to caf, NMDA and BzATP. Incubation of astrocytes with neuron-condition medium (NCM) for 24 h totally abolished the caf-evoked [Ca²⁺]_i increase. In NCM-treated astrocytes, peak of [Ca²⁺]_i rise evoked by NMDA was delayed to about 3.5 min, and that induced by BzATP occurred about three minutes earlier than in SM. The results show that neurons secrete factors that negatively modulate RyR-mediated Ca²⁺-induced Ca²⁺ release (CICR) in astrocytes and alter the time course of Ca²⁺ responses to ionotropic stimuli.     

Sodium-calcium exchange in mammalian heart: current-voltage relation and intracellular calcium concentration

Membrane currents and changes in intracellular calcium ion concentration ([Ca²⁺]_i) have been recorded that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Single guinea-pig ventricular myocytes under voltage clamp were perfused internally with the fluorescent Ca²⁺-indicator, fura-2, and changes in [Ca²⁺]_i and membrane current that resulted from Na-Ca exchange were isolated through the use of various organic channel blockers (verapamil, TTX), impermeant ions (Cs⁺, Ni²⁺), and inhibitors of sarcoplasmic reticulum (ryanodine). The I-V relation of Na-Ca exchange was obtained from the Ni²⁺-sensitive current elicited by ramp repolarization from +90 mV to –80 mV. Ramps were sufficiently rapid that little change in [Ca²⁺]_i occured during the ramp. The (constant) [Ca²⁺]_i during the ramp was varied over the range 100 nM to 1000 nM by varying the amplitude and duration of a pre-pulse to the ramp. The reversal potential of the Ni²⁺-sensitive ramp current varied linearly with 1n([Ca²⁺])_i. The I-V relations at different [Ca²⁺]_i over the range –60 mV to +140 mV were in reasonable accord with the predictions of a simple, simultaneous scheme of Na-Ca exchange, on the basis that only [Ca²⁺]_i had changed. The relationship between [Ca²⁺]_i and current at a constant membrane voltage was also in accord with this scheme. We suggest that Ca²⁺-fluxes through the exchanger during the cardiac action potential can be understood quantitatively by considering the binding of Ca²⁺ to the exchanger during the [Ca²⁺]_i-transient and the effects of membrane voltage on the exchanger.     

Effects of CaMKII-Mediated Phosphorylation of Ryanodine Receptor Type 2 on Islet Calcium Handling,Insulin Secretion,and Glucose Tolerance

Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca²⁺-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) of β cells. This elevation in [Ca²⁺]_cyt leads to activation of Ca²⁺/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca²⁺-release channel implicated in Ca²⁺-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca²⁺ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca²⁺]_cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca²⁺ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.     

Modeling Local X-ROS and Calcium Signaling in the Heart

Stretching single ventricular cardiac myocytes has been shown experimentally to activate transmembrane nicotinamide adenine dinucleotide phosphate oxidase type 2 to produce reactive oxygen species (ROS) and increase the Ca²⁺ spark rate in a process called X-ROS signaling. The increase in Ca²⁺ spark rate is thought to be due to an increase in ryanodine receptor type 2 (RyR2) open probability by direct oxidation of the RyR2 protein complex. In this article, a computational model is used to examine the regulation of ROS and calcium homeostasis by local, subcellular X-ROS signaling and its role in cardiac excitation-contraction coupling. To this end, a four-state RyR2 model was developed that includes an X-ROS-dependent RyR2 mode switch. When activated, [Ca²⁺]_i-sensitive RyR2 open probability increases, and the Ca²⁺ spark rate changes in a manner consistent with experimental observations. This, to our knowledge, new model is used to study the transient effects of diastolic stretching and subsequent ROS production on RyR2 open probability, Ca²⁺ sparks, and the myoplasmic calcium concentration ([Ca²⁺]_i) during excitation-contraction coupling. The model yields several predictions: 1) [ROS] is produced locally near the RyR2 complex during X-ROS signaling and increases by an order of magnitude more than the global ROS signal during myocyte stretching; 2) X-ROS activation just before the action potential, corresponding to ventricular filling during diastole, increases the magnitude of the Ca²⁺ transient; 3) during prolonged stretching, the X-ROS-induced increase in Ca²⁺ spark rate is transient, so that long-sustained stretching does not significantly increase sarcoplasmic reticulum Ca²⁺ leak; and 4) when the chemical reducing capacity of the cell is decreased, activation of X-ROS signaling increases sarcoplasmic reticulum Ca²⁺ leak and contributes to global oxidative stress, thereby increases the possibility of arrhythmia. The model provides quantitative information not currently obtainable through experimental means and thus provides a framework for future X-ROS signaling experiments.     

Mechanistic insights into store-operated Ca2+ entry during excitation-contraction coupling in skeletal muscle

Skeletal muscle fibres support store-operated Ca²⁺-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca²⁺ from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca²⁺]_SR. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca²⁺-release (Communications Biology 1:31, doi: https://doi.org/10.1038/s42003-018-0033-7). Stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel 1 (ORAI1), positioned at the SR and t-system membranes, respectively, are the considered molecular correlate of SOCE. The evidence suggests that at the triads, where the terminal cisternae of the SR sandwich a t-tubule, STIM1 and ORAI1 proteins pre-position to allow for enhanced SOCE transduction.Here we show that phasic SOCE is not only shaped by global [Ca²⁺]_SR but provide evidence for a local activation within nanodomains at the terminal cisternae of the SR. This feature may allow SOCE to modulate [Ca²⁺]_SR during EC coupling. We define SOCE to occur on the same timescale as EC coupling and determine the temporal coherence of SOCE activation to SR Ca²⁺ release. We derive a delay of 0.3 ms reflecting diffusive Ca²⁺-equilibration at the luminal ryanodine receptor 1 (RyR1) channel mouth upon SR Ca²⁺-release. Numerical simulations of Ca²⁺-calsequestrin binding estimates a characteristic diffusion length and confines an upper limit for the spatial distance between STIM1 and RyR1. Experimental evidence for a 4- fold change in t-system Ca²⁺-permeability upon prolonged electrical stimulation in conjunction with numerical simulations of Ca²⁺-STIM1 binding suggests a Ca²⁺ dissociation constant of STIM1 below 0.35 mM. Our results show that phasic SOCE is intimately linked with RyR opening and closing, with only μs delays, because [Ca²⁺] in the terminal cisternae is just above the threshold for Ca²⁺ dissociation from STIM1 under physiological resting conditions.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

α-Parvalbumin reduces depolarizationminduced elevations of cytosolic free calcium in human neuroblastoma cells

We investigated whether the expression of human α-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca²⁺]_i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM.Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K⁺. During this period, [Ca²⁺]_i was monitored by video microfluorimetry using the Ca²⁺ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca²⁺]_i The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca²⁺]_i than non-parvalbumin-expressing cells. Resting [Ca 2+], did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca²⁺]_i in neuroblastoma cells can be suppressed by parvalbumin.     

Intracellular Calcium Homeostasis Changes Induced in Rat Spinal Cord Neurons by Extracellular Acidification

Changes in intracellular Ca²⁺ induced by extracellular acidification to pH = 6 were studied in isolated rat spinal dorsal horn neurons using indo-1 fluorescent technique. In all neurons such treatment induced a decrease of basal [Ca²⁺]_i level by 20.8%, preceded in some of them by temporary increase. The changes were completely reversible. The depolarization-induced [Ca²⁺]_i transients became strongly and also reversibly depressed. If tested after termination of acidification, they demonstrated substantial prolongation of their decay phase, reaching 310% at 120 sec after the application of depolarization. To analyze the mechanisms of such changes, mitochondrial protonophore CCCP has been applied between the end of acidification and the depolarizing pulse. This completely eliminated the described slowing of the transients' decay. To the contrary, application of caffeine to induce Ca²⁺ release from the endoplasmic reticulum did not show significant changes in the corresponding [Ca²⁺]_i transients. A conclusion is made that in mammalian neurons extracellular acidification, apart from inhibiting voltage-operated Ca²⁺ channels, also substantially alters the Ca²⁺ exchange function of mitochondria responsible for rapid accumulation of ions and their delayed release back into the cytosol.     

Functional abnormalities in iPSC‐derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca²⁺ handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation‐contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca²⁺ abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell‐derived cardiomyocytes (iPSC‐CM) from CPVT1 and CPVT2 patients carrying the RyR2^R420Q and CASQ2^D307H mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC‐CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca²⁺]_i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC‐CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca²⁺]_i. Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC‐CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.     

Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload

Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca²⁺-handling. We investigated the effects of ascending aortic banding (AoB) on Ca²⁺-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca²⁺-release at the cell edge whereas Ca²⁺ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na⁺/Ca²⁺ exchanger, SR Ca²⁺ ATPase or phospholamban. Mathematical modeling indicated that [Ca²⁺]_i transients at the cell center were accounted for by simple centripetal diffusion of Ca²⁺ released at the cell edge. In contrast, caffeine (10 mM) induced Ca²⁺ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca²⁺-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca²⁺ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca²⁺-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca²⁺-release.     

Membrane topology and membrane retention of the ryanodine receptor calcium release channel

The ryanodine receptor (RyR) is a Ca²⁺ release channel located in the sarcoplasmic/endoplasmic reticulum (ER) membrane and plays a critical role in excitation-contraction coupling of skeletal and cardiac muscles. RyR normally exists in a tetrameric structure and contains two functional domains: a carboxyl-terminal hydrophobic domain that contains the conduction pore of the Ca²⁺ release channel, and a large amino-terminal domain that contains sites responsible for channel regulation. Recent studies involving mutagenesis and heterologous expression have helped unravel the structure-function relationship of RyR, including transmembrane topology and intracellular localization of the Ca²⁺-release channel. The carboxyl-terminal portion of RyR contains the putative transmembrane segments and is sufficient to form a functional Ca²⁺-release channel. The amino-terminal region of the protein contains sites responsible for regulation by endogenous modulators such as Ca²⁺ and Mg²⁺ and by exogenous ligands such as caffeine. The membrane topology of RyR appears to contain an even number (four or six) of transmembrane segments with a ion selectivity filter present within a region residing between the last two segments, similar to potassium channel, whose atomic structure was described recently. The transmembrane segments also contain sequences that are responsible for localization of RyR in the endoplasmic reticulum, and this sequence is highly conserved in IP₃ receptors, which also function as Ca²⁺-release channels.