Calcium-induced Release Research Articles

MiR-25 in heart failure.

Recent work by Wahlquist et al 1 showed miR-25 to reduce levels of rco/endoplasmic reticulum calcium-ATPase (SERCA) 2a during heart failure, whereas therapeutic inhibition of this microRNA (miRNA) was able to reverse pre-established heart failure in a mouse model of pressure overload. Although these data suggest that miR-25 might be a drug target for the treatment of heart failure, further in vivo analysis is required before we can consider miR-25 inhibition for clinical application. Recently, Wahlquist et al1 reported on the pathological upregulation of miR-25 during heart failure and showed that inhibition was able to block and reverse the disease in mice. Although an increase in cardiac miR-25 levels caused a decline in cardiac function, anti–miR-based inhibition of miR-25 halted established heart failure at least in part by increasing the mRNA of SERCA2a. This is intriguing because phase II clinical data from the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial has demonstrated that gene therapy to increase SERCA2a levels in humans delays the progression of chronic heart failure. This could imply that pharmacological regulation of miR-25 may regulate at least 1 clinically validated target, which may be more straightforward than current gene therapy approaches. However, miRNA biology seems to sketch a somewhat more complex picture than a miRNA functioning through 1 single-gene target. In addition, conflicting data on the cardiac functions of miR-25 highlight the need for further investigation before advancing miR-25 as a clinical target for heart failure. miRNAs are short single-stranded noncoding RNAs that modulate the expression of target proteins by annealing to complementary sequences in their target mRNAs.2 It is thought that a single vertebrate miRNA on average targets a few hundred transcripts.3,4 Because individual miRNAs often target numerous related mRNAs that encode multiple components of …

Differential effect of calcium-activated potassium and chloride channels on rat basilar artery vasomotion.

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. We investigated the cellular mechanisms underlying vasomotion in the cerebral basilar artery (BA) of Wistar rats. Pressure myograph video microscopy was used to study the changes in cerebral artery vessel diameter. The main results of this study were as follows: (1) The diameters of BA and middle cerebral artery (MCA) were 314.5±15.7 μm (n=15) and 233.3±10.1 μm (n=12) at 10 mmHg working pressure (P<0.05), respectively. Pressure-induced vasomotion occurred in BA (22/28, 78.6%), but not in MCA (4/31, 12.9%) from 0 to 70 mmHg working pressure. As is typical for vasomotion, the contractile phase of the response was more rapid than the relaxation phase; (2) The frequency of vasomotion response and the diameter were gradually increased in BA from 0 to 70 mmHg working pressure. The amplitude of the rhythmic contractions was relatively constant once stable conditions were achieved. The frequency of contractions was variable and the highest value was 16.7±4.7 (n=13) per 10 min at 60 mmHg working pressure; (3) The pressure-induced vasomotion of the isolated BA was attenuated by nifedipine, NFA, 18β-GA, TEA or in Ca(2+)-free medium. Nifedipine, NFA, 18β-GA or Ca(2+)-free medium not only dampened vasomotion, but also kept BA in relaxation state. In contrasts, TEA kept BA in contraction state. These results suggest that the pressure-induced vasomotion of the isolated BA results from an interaction between Ca(2+)-activated Cl(-) channels (CaCCs) currents and K(Ca) currents. We hypothesize that vasomotion of BA depends on the depolarizing of the vascular smooth muscle cells (VSMCs) to activate CaCCs. Depolarization in turn activates voltage-dependent Ca(2+) channels, synchronizing contractions of adjacent cells through influx of extracellular calcium and the flow of calcium through gap junctions. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates K(Ca) channels and hyperpolarizes VSMCs, which provides a negative feedback loop for regenerating the contractile cycle.

Modelling mechanism of calcium oscillations in pancreatic acinar cells.

We present a simple model for calcium oscillations in the pancreatic acinar cells. This model is based on the calcium release from two receptors, inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) through the process of calcium induced calcium release (CICR). In pancreatic acinar cells, when the Ca²⁺ concentration increases, the mitochondria uptake it very fast to restrict Ca(2+) response in the cell. Afterwards, a much slower release of Ca²⁺ from the mitochondria serves as a calcium supply in the cytosol which causes calcium oscillations. In this paper we discuss a possible mechanism for calcium oscillations based on the interplay among the three calcium stores in the cell: the endoplasmic reticulum (ER), mitochondria and cytosol. Our model predicts that calcium shuttling between ER and mitochondria is a pacemaker role in the generation of Ca²⁺ oscillations. We also consider the calcium dependent production and degradation of (1,4,5) inositol-trisphosphate (IP3), which is a key source of intracellular calcium oscillations in pancreatic acinar cells. In this study we are able to predict the different patterns of calcium oscillations in the cell from sinusoidal to raised-baseline, high frequency and low-frequency baseline spiking.

A method for multi-simulator reaction-diffusion with NEURON

There are two major approaches in the development of simulation tools for multi-scale modeling – the all-in-one simulator; and the unifying simulation environment that allows multiple plug-ins to be utilized in order to provide different aspects of a simulation. While NEURON has traditionally taken the former approach, it has become clear that there are always going to be multiple approaches even to modeling at a single level, making it desirable to be able to readily plugin in external simulators to provide improvements or to try comparisons. For example, at the level of reaction-diffusion, the built-in scheme provides techniques that differ somewhat from those of Virtual Cell [1] or XTW [2], both of which are now available as plugin environments. NEURON is a widely used simulator for biophysically detailed neuronal electrophysiology models that has recently been extended with syntax for reaction-diffusion dynamics [3]. This class of problems is useful for studying the intracellular dynamics of neurons – e.g. protein-protein interactions or calcium-induced calcium release (CICR) – and how they interact with the electrophysiology. As many molecules play a role in both intracellular dynamics and electrophysiology, these must be simulated simultaneously. The developmental branch of NEURON offers experimental 3D reaction-diffusion support, but its performance remains below that of dedicated reaction-diffusion simulators. To leap-frog over these performance issues, we have developed a new method for multi-simulator studies. In our technique, all model specification and simulation protocols remain specified in NEURON. Thus, switching between NEURON’s experimental integrated 3D solver and an external solver requires only adding the connection code. No familiarity with the external solver is required in contrast to multi-simulations with MUSIC [4], where the models are described using each simulator’ sn ative description. The simulators run in different processes. All model specification, currents, and concentration information is exchanged via shared memory. As an example, we demonstrate using our technique to study the behavior of CICR waves near the soma of a pyramidal neuron.

Integrating Systems Biology Markup Language(SBML) with NEURON

The NEURON simulator software is widely used by the computational neuroscience community for neuronal electrophysiology modeling and we have recently extended it to support reaction-diffusion dynamics [1]. This class of dynamics is ideal for studying intracellular dynamics, such as calcium-induced calcium release. NEURON’s original reaction-diffusion support introduced a custom syntax; although this allowed taking advantage of NEURON’s full capabilities, it required existing cell biology models to be rewritten. To eliminate this barrier, we have introduced SBML support for NEURON. SBML [2] is an XML-based representation format used for specifying computational models of biological processes. This format is one of the most widely used, and is supported by over 200 software packages. It has become a standard used by systems biologists for the exchange of information about chemical reaction dynamics. We have introduced SBML support to the reaction diffusion module of the NEURON simulation software. This enables NEURON users to import a large number of previously developed cell biology models and use them in computational neuroscience research. We have added the capability to import and export models in SBML format to NEURON’s reaction-diffusion module using the libSBML library [3]. This allows one to combine models from ModelDB [4] with cell models from the BioModels database [5] in a way that makes it possible to match state variables and to readily change parameters. The procedure is as follows: • Electrophysiology model is loaded from ModelDB or constructed de novo. • NEURON loads SBML data and instantiates appropriate reaction diffusion objects: rxd.Region, rxd.Species, rxd.Reaction. User must interactively or preemptively match state-variable names across combined models to combine the two models into one. • SBML models do not include diffusion so diffusion constants can be added to provide the spatial element of the combined model. • User can adjust the parameters and make simulation runs of the model. We have used these new features to study the interaction of a calcium induced calcium release model from BioModels with electrical activity in a detailed model of a hippocampal CA1 pyramidal neuron.

The self-regulation of neurotransmitter release.

Retzius cells are large neurons in the ganglia of the leech (Lent, 1977), releasing 5-HT (Leake, 1986) and orchestrating the animal behavior (Carretta, 1988). The leech nervous system is relatively simple and its exploration allows correlating, in a quantitative manner, the chemical and electrical activity, at cellular level, with the animal behavior (Mazzoni et al., 2007). The recent work of Francisco De-Miguel, Citlali Trueta and colleagues has given important contributions on the understanding of 5-HT secretion at Retzius cells that occurs both at the site of synaptic targets and from the soma (De-Miguel and Trueta, 2005). The somatic 5-HT release is dependent on L-type calcium channel activation (Trueta et al., 2003) and on calcium-induced calcium release (Trueta et al., 2004). Retzius cells have 5-HT autoreceptors and the activation of these proteins is coupled to chloride channels producing synaptic autoinhibition (Cercos et al., 2009). In the article by Leon-Pinzon et al. (2014) recently published in Frontiers in Cellular Neuroscience, a significant progress on the understanding of the mechanisms induced by 5-HT autoreceptors is presented. Specifically, the authors report the very interesting finding that sustained 5-HT vesicular release is regulated by a positive feedback at the same Retzius cell. The soma of Retzius neurons releases serotonin from dense clusters of core vesicles without evidence of active zones. This type of secretion is similar to secretion by excitable endocrine cells. When 5-HT release is associated with high-frequency action potential firing, the initial 5-HT release mediated by calcium entry via voltage-gated calcium channels activates 5-HT autoreceptors. This activation leads to one or two additional episodes of intracellular calcium increase which amplify the release of 5-HT. In order to perform this study, the authors used an elegant experimental analysis combining electrophysiological recordings, fluorescence imaging of calcium and neurotransmitter activity and pharmacological tests. The physiological amplification mechanism reported in this paper allows Retzius cells operating in a two-state mode: a “weakly releasing” mode and a “strongly releasing” mode. Presumably, there will be a critical frequency that may vary from cell to cell switching from one mode to the other. This bi-stable behavior, which might be similar to what reported in another invertebrate neuron (Achenbach et al., 1997), plays an obviously important role in the computational properties of both Retzius cells and associated neuronal networks. But did we learn something that can be possibly extended to the mammalian nervous system? The leech nervous system represents a rich model for vertebrate systems from the points of view of both cellular neurobiology (Schmold and Syed, 2012) and of higher functions (Sahley, 1995). In serotonergic neurons in the raphe nuclei of the rodent brain, 5-HT is also released from the soma (Sarkar et al., 2012) and from the dendrites (Colgan et al., 2012). These phenomena have several common features with 5-HT somatic release at Retzius neurons. Thus, Leon-Pinzon et al. (2014) formulate the hypothesis that the mechanisms of self-regulation of 5-HT discovered in Retzius neurons may also operate in the mammalian nervous system, perhaps by activating receptors not only at the soma and at different time-scale.

P134Increased beta-adrenergic inotropy in ventricular myocardium from Trpm4 knockout mice

TRPM4 have an extensive role in regulating intracellular calcium dependent cell functions in many cells including mast cells, chromaffin cells and recently in neurons. TRPM4 proteins constitute calcium activated, but calcium impermeable, non-selective cation channels and are expressed both in atrial and ventricular cardiomyocytes. The Trpm4 gene has recently been associated with several disorders, including cardiac conduction diseases such as Progressive Familial Heart Block Type 1, Right Bundle Branch Block and Brugada Syndrome. The physiological function of TRPM4 in the heart remains however incompletely understood. The objective of this study was to establish the role of TRPM4 in left ventricular muscle function. We used TRPM4 knockout mice and performed patch-clamp experiments, membrane potential measurements, microfluorometry, contractility measurements and in vivo pressure-volume loop analysis. We demonstrate that TRPM4 proteins are functionally present in mouse ventricular myocytes and are activated upon calcium induced calcium release. Measurements of action potential duration show that TRPM4 is active during the repolarization of the ventricular action potential. We show a significantly decreased time for 50 % and 90 % repolarization in Trpm4 knockout ventricular myocytes. We also provide evidence that this change in action potential shape leads to an increased driving force for the L-type calcium current during the action potential. Moreover, in vivo pressure-volume analysis and in vitro contraction strength measurement on isolated papillary muscles displays an increased β-adrenergic inotropic response in TRPM4 knockout mice. Taking together these data, our results show that functional TRPM4 proteins are novel determinants of the inotropic effect of β-adrenergic stimulation on the ventricular heart muscle.

Role of intracellular calcium stores in hair-cell ribbon synapse

Intracellular calcium stores control many neuronal functions such as excitability, gene expression, synaptic plasticity, and synaptic release. Although the existence of calcium stores along with calcium-induced calcium release (CICR) has been demonstrated in conventional and ribbon synapses, functional significance and the cellular mechanisms underlying this role remains unclear. This review summarizes recent experimental evidence identifying contribution of CICR to synaptic transmission and synaptic plasticity in the CNS, retina and inner ear. In addition, the potential role of CICR in the recruitment of vesicles to releasable pools in hair-cell ribbon synapses will be specifically discussed.

Mitochondrial Hsp90s suppress calcium-mediated stress signals propagating from mitochondria to the ER in cancer cells.

BackgroundResistance to cell death in the presence of stressful stimuli is one of the hallmarks of cancer cells acquired during multistep tumorigenesis, and knowledge of the molecular mechanism of stress adaptation can be exploited to develop cancer-selective therapeutics. Mitochondria and the endoplasmic reticulum (ER) are physically interconnected organelles that can sense and exchange various stress signals. Although there have been many studies on stress propagation from the ER to mitochondria, reverse stress signals originating from mitochondria have not been well reported.MethodsAfter inactivation of the proteins by pharmacologic and genetic methods, the signal pathways were analyzed by fluorescence microscopy, flow cytometry, MTT assay, and western blotting. A mouse xenograft model was used to examine synergistic anticancer activity and the action mechanism of drugs in vivo.ResultsWe show in this study that mitochondrial heat shock protein 90 (Hsp90) suppresses mitochondria-initiated calcium-mediated stress signals propagating into the ER in cancer cells. Mitochondrial Hsp90 inhibition triggers the calcium signal by opening the mitochondrial permeability transition pore and, in turn, the ER ryanodine receptor, via calcium-induced calcium release. Subsequent depletion of ER calcium activates unfolded protein responses in the ER lumen, thereby increasing the expression of a pro-apoptotic transcription factor, CEBP homologous protein (CHOP). Combined treatment with the ER stressor thapsigargin and the mitochondrial Hsp90 inhibitor gamitrinib augmented interorganelle stress signaling by elevating CHOP expression, and showed synergistic cytotoxic activity exclusively in cancer cells in vitro and in vivo.ConclusionsCollectively, mitochondrial Hsp90s confer cell death resistance to cancer cells by suppressing the mitochondria-initiated calcium-mediated interorganelle stress response.

Translating Intracellular Calcium Signaling into Models

The rich experimental data on intracellular calcium has put theoreticians in an ideal position to derive models of intracellular calcium signaling. Over the last 25 years, a large number of modeling frameworks have been suggested. Here, I will review some of the milestones of intracellular calcium modeling with a special emphasis on calcium-induced calcium release (CICR) through inositol-1,4,5-trisphosphate and ryanodine receptors. I will highlight key features of CICR and how they are represented in models as well as the challenges that theoreticians face when translating our current understanding of calcium signals into equations. The selected examples demonstrate that a successful model provides mechanistic insights into the molecular machinery of the Ca²⁺ signaling toolbox and determines the contribution of local Ca²⁺ release to global Ca²⁺ patterns, which at the moment cannot be resolved experimentally.

Modelling the transition from simple to complex Ca²⁺ oscillations in pancreatic acinar cells.

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP3) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca2+ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP3 is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.

Steven Houser: the beat goes on.

During 36 years at Temple University in Philadelphia, Steven Houser has been a leader in advancing scientific understanding of heart function. All along, he’s been driven by a desire to understand his father’s fatal heart disease, and to contribute important insights to help future patients. Steven Houser Houser and his collaborators—along with others in the field, he is quick to note—have shed light on numerous fundamental questions about how calcium regulates the beating of cardiomyocytes, including elucidating the details of calcium-induced calcium release.1,2 His research is probing calcium-dependent signaling pathways, aberrations in the hypertrophic, failing, or post-myocardial infarction heart,3–6 factors related to myocyte turnover,7 and potential treatment strategies including gene and stem cell therapy. Houser’s lab recently demonstrated that cortical bone-derived stem cells may be superior to cardiac stem cells in regenerating heart tissue after MI.8 Houser, 63, director of the Cardiovascular Research Center and chair of physiology at Temple, tells Circulation Research he’s never afraid to jump into a scientific argument9 and help inform the debate. His strategy is always fair and careful experimentation, with an emphasis on reproducible findings. “I think the results from my group have stood the test of time,” he says. “That’s one of the things I’m proudest of—that our work can be repeated.” I grew up in New Jersey, in a little town called Magnolia, about 10 miles from Philadelphia, in suburban housing built primarily for soldiers returning after World War II. My dad had been in the service. [He] was a working guy. Mom stayed home, tried to keep me in line. My dad worked for a company called Western Electric. He was a wireman. My mom ended up going to college when I started high school. To help pay my way through college, …

Effects of eugenol on resting tension of rat atria

In cardiac and skeletal muscle, eugenol (μM range) blocks excitation-contraction coupling. In skeletal muscle, however, larger doses of eugenol (mM range) induce calcium release from the sarcoplasmic reticulum. The effects of eugenol are therefore dependent on its concentration. In this study, we evaluated the effects of eugenol on the contractility of isolated, quiescent atrial trabeculae from male Wistar rats (250-300 g; n=131) and measured atrial ATP content. Eugenol (1, 3, 5, 7, and 10 mM) increased resting tension in a dose-dependent manner. Ryanodine [100 µM; a specific ryanodine receptor (RyR) blocker] and procaine (30 mM; a nonspecific RyR blocker) did not block the increased resting tension induced by eugenol regardless of whether extracellular calcium was present. The myosin-specific inhibitor 2,3-butanedione monoxime (BDM), however, reversed the increase in resting tension induced by eugenol. In Triton-skinned atrial trabeculae, in which all membranes were solubilized, eugenol did not change resting tension, maximum force produced, or the force vs pCa relationship (pCa=-log [Ca2+]). Given that eugenol reduced ATP concentration, the increase in resting tension observed in this study may have resulted from cooperative activation of cardiac thin filaments by strongly attached cross-bridges (rigor state).

Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle

The Ca(2+) uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca(2+)] heavily buffered in the pCa range (-log10[Ca(2+)]) 7.3-6.0 for set times and the amount of net SR Ca(2+) accumulation determined from the force response elicited upon emptying the SR of all Ca(2+). Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.6. Maximal Ca(2+) uptake rate was ∼0.18 and ∼0.21 mmol Ca(2+) (l fibre)(-1) s(-1) in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg(2+)] from 1 to 3 mM had no significant effect on the net Ca(2+) uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca(2+) release channels during uptake in either fibre type. Ca(2+) leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca(2+) leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca(2+) uptake in the type I human fibres can account for the higher relative level of SR Ca(2+) loading observed in type I compared to type II fibres, and the SR Ca(2+) leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.

Effect of Different Currents and Extracellular Potassium Ion Concentration on Anodal Excitation of Cardiac Tissue

Background:In order to understand defibrillation completely and thereby to design better defibrillators, we need to understand fibrillation and the effect of electrical stimulation on cardiac tissue. We address this issue by studying anodal excitation applied to the refractory tissue and by measuring the refractoriness of the cardiac tissue through the strength-Interval (SI) curve. The anodal SI curve contains a in which the S2 threshold increases as the interval increases. Our goal is to find the mechanism of the dip and to determine how calcium currents, sodium calcium exchange (NCX) current and elevated extracellular potassium ion concentration, [K]e, influences the dip in the anodal strength-interval curve.Methods and Results:Computer simulations of unipolar stimulation were performed using the bidomain model, with membrane kinetics governed by the Luo-Rudy model. The SI curve is determined by applying a threshold stimulus at different time intervals after a previous action potential. The dip disappears with no NCX current, but is present with 50% and 75% reduction of normal NCX current. The calcium induced calcium release (CICR) current and/or calcium uptake current are not responsible for the dip in the anodal SI curve. High [K]e results in the disappearance of the dip in the anodal SI curve because the tissue remained refractory after the transmembrane potential returned to its resting state.Conclusions:Neither NCX nor calcium current is responsible for the dip in the anodal SI curve. It is due to the electrotonic interaction between regions of depolarization and hyperpolarization following the S2 stimulus. The dominance of the electrotonic mechanism emphasizes the importance of the spatial distribution of virtual electrodes during excitation, which ultimately clarifies tissue-shock interactions and optimizes advanced defibrillation protocols.

Presenilins: A novel class of calcium modulators

A disease (AD) is the most common form of dementia, affecting more than 5 mi Americans. As such, AD poses a significant burden on the affected individual, caregivers and society. Most cases of AD are attributed to the sporadic form, which is believed to be of multifactorial origin. However, several genetic loci etiological for the rare familial form of the disease have been identified. One of the loci is the group of presenilin proteins, which form the enzymatic core of the γ-secretase complex. Most of the almost 200 identified familial AD mutations in presenilins are located in the gene encoding presenilin-1, while presenilin-2 mutations typically cause later onset familial AD. Recent evidence identified the group of presenilin proteins as potent modulators of intracellular calcium signaling, through potentiation of the intracellular ryanodine receptor, which likely underlies this phenomenon. This potentiation occurs via the highly evolutionarily conserved N-terminal region of presenilin, resulting in differential modulation of the ryanodine receptor by presenilin-1 and presenilin-2. The proposed mechanism is in accordance with previous studies identifying elevated Ca2+ concentrations in the endoplasmic reticulum during AD, and the critical role of ryanodine receptors in regulating calcium via calcium-induced calcium release. Furthermore, ryanodine receptors contribute to the pathologic, elevated intracellular Ca2+ concentrations observed in AD. Intriguingly, similar Ca2+ dyshomeostasis occurs during healthy aging, in the absence of known mutations. Utilizing preclinical models for healthy aging, we have implicated presenilin proteins in the etiology of age-related changes in synaptic signaling and, ultimately, agerelated deficits in memory and motor coordination. In this keynote talk, the author will summarize the evidence for the group of presenilin proteins as a novel class of calcium modulators, and discuss the opportunities for targeting presenilin proteins as novel drug targets for age-related and neurodegenerative diseases, incl. AD.

Dynamics of Calcium Sparks and SR Calcium Leak During Excitation-Contraction Coupling in Mouse Heart Cells

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cell contraction during cardiac excitation-contraction (EC) coupling in heart cells. The fundamental element of CICR in heart is the calcium (Ca2+) spark which arises from the regenerative release of Ca2+ from a cluster of ryanodine receptors (RyR2) in the junctional sarcoplasmic reticulum membrane. We have shown how stochastic gating of RyR2s could produce an SR Ca2+ leak capable of balancing SR Ca2+-ATPase (SERCA2a) activity under quiescent conditions (Williams et al. BJ 2011). This investigation suggested the surprising finding that a single, or even multiple, RyR2 openings could fail probabilistically to trigger a Ca2+ spark. To further investigate this effect, we expanded upon that formulation to create a detailed, local control model of EC coupling in mouse heart. A number of features were added or modified, including the addition of a novel seven-state Markov chain model of the sarcolemmal L-type Ca2+ channel (LCC). This model features dynamic action-potential (AP) generation, robust Ca2+ spark initiation and termination, realistic [Ca2+]i transients, and true SR Ca2+ pump/leak balance. The model suggests that numerous LCC openings are often required to trigger a single Ca2+ spark. This is consistent with our prior work where multiple RyR2 openings were needed to trigger a spontaneous Ca2+ spark during quiescent conditions. We also investigated how RyR2 mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) influence the dynamics of Ca2+ sparks, “invisible” non-spark Ca2+ leak, [Ca2+]i transients, and SR Ca2+ content. We observe that CPVT mutations can lead to unstable Ca2+ spark dynamics, altering SR Ca2+ content and promoting [Ca2+]i signaling instability. Our new model provides significant insights into the dynamics of local control of CICR under physiological and pathological conditions.

Calcium-induced calcium release mediates all-or-nothing responses of mesenchymal stromal cells to noradrenaline

By using Ca2+ imaging and Fluo-4 dye, we examined the capability of certain agonists of G-protein coupled receptors to stimulate Ca2+ signaling in cultured mesenchymal stromal cells (MSC) derived from the human adipose tissue. In particular, a small subpopulation (∼5%) MSC was found to respond to noradrenaline with Ca2+ transients. The all-or-nothing fashion was characteristic of adrenergic Ca2+ signaling in MSC, that is, while at low concentrations noradrenaline stimulated undetectable Ca2+ transients, virtually maximal responses were elicited by this agonist at any concentration above the threshold of 100–200 nM. In some experiments, MSC were loaded with the photosensitive Ca2+ chelator NP-EGTA to produce local or global jumps in cytosolic Ca2+ concentration by virtue of Ca2+ uncaging. Global uncaging eliciting a high enough Ca2+ jump triggered a Ca2+ transient in the MSC cytoplasm, which was similar to a noradrenaline response kinetically and by magnitude. When Ca2+ uncaging was produced locally, it initiated a Ca2+ signal that traveled along a cell with a speed that exceeded an expected one by two orders of magnitude, should Ca2+ signal transfer be mediated merely by passive Ca2+ diffusion in the presence of Ca2+ buffer. These findings implicated Ca2+-induced Ca2+ release (CICR) as a mechanism amplifying local Ca2+ signals in MSC. Of Ca2+ targets involved in CICR, the ryanodine receptor and IP3 receptor are only known. The inhibitory analysis revealed IP3 receptors to be principally responsible for CICR in MSC, whereas a contribution of ryanodine receptors was negligible. Altogether, our results suggest that an initial noradrenaline-dependent rise in cytosolic Ca2+ stimulates, should it reach the threshold level, IP3 receptors, thereby triggering an avalanche-like Ca2+ release from Ca2+ stores and underlying the all-or-nothing dependence of cellular responses on the agonist concentration.

Examination of Single Channel RyR Behavior from Long-Lasting Ca2+ Sparks

Calcium sparks are crucial to cardiac excitation-contraction coupling and are due to calcium-induced calcium release via ryanodine receptors (RyRs) located on the sarcoplasmic reticulum (SR). A calcium spark results from the concerted opening of a cluster of RyRs, which generally closes by a robust mechanism as shown by stereotypic spark duration (∼40 ms). However, much longer calcium sparks (up to seconds) can be seen in cells isolated from an animal models of heart failure (1) and when RyRs have been pharmacologically inhibited (2). The mechanism underlying such long-lasting events is unclear. It has been suggested that a lack of local SR calcium depletion (from a combination of decreased release flux and increased SR calcium buffering and/or refilling) is important (2), however it has also been suggested that the activity of RyR sub-clusters (located 1 ms =∼1 um apart) may also sustain release (1). We examined calcium sparks in cells treated with tetracaine to partially inhibit RyRs. Spark-like and long-lasting events occurred at the same location in a line-scan image, suggesting a single release site is able to exhibit both types of behavior. Analysis of long-lasting release events (mean duration = 315 ms) yielded results consistent with a mechanism that involves a reduction in RyR availability and there was no detectable change in the location of the origin of release. These conclusions are supported by a computer model that was able to reproduce long-lasting events when the number of stochastically gating RyRs available was reduced and SR calcium was increased. Large alterations in RyR cluster geometry were not required, but the geometric organization of RyRs in the cluster plays an important role.

The coupling of plasma membrane calcium entry to calcium uptake by endoplasmic reticulum and mitochondria

Cross-talk between organelles and plasma membrane Ca(2+) channels is essential for modulation of the cytosolic Ca(2+) ([Ca(2+)]C) signals, but such modulation may differ among cells. In chromaffin cells Ca(2+) entry through voltage-operated channels induces calcium release from the endoplasmic reticulum (ER) that amplifies the signal. [Ca(2+)]C microdomains as high as 20-50 μm are sensed by subplasmalemmal mitochondria, which accumulate large amounts of Ca(2+) through the mitochondrial Ca(2+) uniporter (MCU). Mitochondria confine the high-Ca(2+) microdomains (HCMDs) to beneath the plasma membrane, where exocytosis of secretory vesicles happens. Cell core [Ca(2+)]C is much smaller (1-2 μm). By acting as a Ca(2+) sink, mitochondria stabilise the HCMD in space and time. In non-excitable HEK293 cells, activation of store-operated Ca(2+) entry, triggered by ER Ca(2+) emptying, also generated subplasmalemmal HCMDs, but, in this case, most of the Ca(2+) was taken up by the ER rather than by mitochondria. The smaller size of the [Ca(2+)]C peak in this case (about 2 μm) may contribute to this outcome, as the sarco-endoplasmic reticulum Ca(2+) ATPase has much higher Ca(2+) affinity than MCU. It is also possible that the relative positioning of organelles, channels and effectors, as well as cytoskeleton and accessory proteins plays an important role.