Intracellular Ca Dynamics Research Articles

Modulating mitochondrial phosphate transport shapes intracellular Ca signaling to impact arrhythmogenesis.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondria Ca uptake and buffering. Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knock out (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. An inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Our results suggest that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce Ca waves and cardiac arrhythmias in CPVT.

Abstract 13377: Modulating Mitochondrial Phosphate Transport Shapes Intracellular Ca Signaling to Impact Arrhythmogenesis

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. Hypothesis: We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondrial Ca uptake and buffering. Methods: Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knockout (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. Results: In the presence of β agonist isoproterenol, an inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Conclusions: Modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce arrhythmogenic Ca waves and cardiac arrhythmias in CPVT.

The increase of extracellular Ca2+ from physiological concentrations to hypercalcemia impairs sino-atrial automaticity

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): ESC FRM Lefoulon Delalande Aims To investigate whether extracellular hypercalcemia alters the conduction through L-type Ca2+ channels (LTCCs), impairing the pacemaker activity of the heart. Introduction In the sino-atrial node (SAN), membrane currents and the dynamics of intracellular Ca2+ ([Ca2+]i) generate the pacemaker activity of the heart. SAN dysfunctions (SNDs) harm heart automaticity and have been associated with abnormal dynamics of [Ca2+]i. The LTCCs, Cav1.2 and Cav1.3 carry the main Ca2+ influx of SAN cells, which is necessary to sustain [Ca2+]i dynamics. Modified extracellular Ca2+ ([Ca2+]o) could alter Ca2+ influx through these channels. For example, cancer and hyperparathyroidism can raise [Ca2+]o, causing an extracellular hypercalcemia that could alter [Ca2+]i dynamics and impair SAN activity and heart automaticity. Methods and results To test this hypothesis, we measured contractions, [Ca2+]i release and L-type Ca2+ current (ICa,L) in spontaneous cells of the murine SAN. Then, we recorded rate and propagation of the spontaneous action potentials (APs) generated by the SAN tissue ex-vivo. In spontaneously beating SAN cells, we observed that the modification of [Ca2+]o affected [Ca2+]i and cell contractility through changes of ICa,L. In particular, the increase of [Ca2+]o dysregulated pacemaker activity, likely through excessive Ca2+ influx mediated by Cav1.2. [Ca2+]o increase to hypercalcemia induced arrhythmia also in the intact SAN tissues, activating ectopic leading regions of pacemaking and impairing conduction towards the atria. Conclusions Hypercalcemia causes excessive Cav1.2-mediated Ca2+ influx, which alters [Ca2+]I leading to pacemaker impairment. Modulation of LTCC may reduce pacemaker dysfunctions, preventing SND progression.

The Mechanism Of Action Of Polyphenol On Changes In The Dynamics Of Calcium In The Synaptosomes Of The Rat Brain Against The Background Of Glutamate

The manuscript shows a short data used using fluorescent probes to study the effect of polyphenol PС-7 on changes in the dynamics of intracellular Ca2+ content in rat brain synaptosomes, depending on the site of glutamate binding on calcium channels by a specific mediator with glutamate. To measure the amount of cytosolic Ca2+ synaptosomes, we calculated using the Grinkevich equation. It has been shown that polyphenol PС-7 binds to the β1-subunit of the voltage-gated calcium channel and allosterically changes its conformation so that the conductivity for Ca2+ ions increases through the channel, the blocking effect of polyphenol PС-7 can be explained by its binding to voltage-gated calcium channels and activating them.

Dynamics of calcium signals and the intracellular regulatory signaling network

<p indent="0mm">The calcium ion (Ca²⁺) acts as a ubiquitous cellular messenger, regulating a wide variety of cellular processes. The intracellular signaling network precisely controls the normal and pathophysiological functions of cells to conduct various physiological activities. This article reviews our research progress on the dynamics of intracellular Ca²⁺ signaling and cellular signaling network models in recent years, including the release of local Ca²⁺ signals from clustered Ca²⁺ channels, global Ca²⁺ waves in cells, Ca²⁺ regulation with microdomains between the endoplasmic reticulum and mitochondria, dynamics of cell apoptosis regulated by Ca²⁺ signaling, and dynamics of cellular signaling regulation networks. These simulation studies provide directions and ideas for future research on how Ca²⁺ signals and cellular signaling networks regulate the complicated life functions in cells.

Suppression of ventricular arrhythmia by mitochondrial calcium uptake via mitochondrial calcium uniporter in the ischemic heart failure mice

Abstract Background In heart failure (HF), diastolic calcium (Ca) leak from sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) causes delayed after depolarization (DAD), leading ventricular arrhythmias (VAs). Recent study reported that Ca uptake into mitochondria via mitochondrial calcium uniporter (MCU) suppress Ca waves (CaWs) and DAD in catecholaminergic polymorphic ventricular tachycardia, in which diastolic Ca leak is thought to be a major cause of VAs as in HF. However, such anti-arrhythmic effect of mitochondrial Ca uptake via MCU remains unclear in HF. Purpose We sought to investigate whether mitochondrial Ca uptake via MCU decreases CAWs and VAs incidence in ischemic HF mice. Methods Ten-week-old male C57BL/6J mice were divided into 2 groups; sham operation mice (Sham) or HF mice (HF) in which myocardial infarction was induced by left coronary artery ligation. After 4–6 weeks, cardiomyocyte or mitochondria was isolated respectively from the myocardium of Sham and the non-infarct myocardium of HF. Influence of MCU activation on Ca dynamics, VA inducibility and left ventricular hemodynamics were evaluated using Kaemenpferol, a MCU activator. Intracellular Ca dynamics and mitochondrial Ca uptake were measured in isolated cardiomyocytes loaded with Fluo-4 AM on an epifluorescence microscopy and by estimating the extra-mitochondrial Ca reduction with Fluo-5N on a spectrofluoro-photometer, respectively. VAs was induced by programmed stimulation in the Langendorff perfused hearts. Left ventricular (LV) pressure was measured using a microtip transducer catheter. Finally, the effect of intravenous administration of Kaempferol (5mg/kg) on hemodynamic parameters was examined 30 minutes after administration in Sham and HF. Results HF mice showed left ventricular dysfunction, as well as the increased heart and lung weights compared to Sham. MCU protein expression in cardiomyocytes did not differ between Sham and HF. Kaempferol increased mitochondrial Ca uptake in the isolated mitochondria both in Sham and HF. The number of the diastolic CaWs was higher in HF compared to Sham. Such increased number of CaWs in HF was attenuated by 10 μM Kaempferol, which was, however, abolished by a MCU blocker Ruthenium Red. The incidence of induced VA was significantly higher in HF than Sham, which was suppressed by Kaempferol. In vivo measurements, intravenous administration of Kaempferol did not show significant changes in hemodynamic parameters in Sham and HF mice. Conclusions Mitochondrial Ca uptake via MCU suppresses CaWs and VAs, but did not change LV hemodynamics in HF. Whereas traditional antiarrhythmic drugs have limited use in heart failure patients, a novel strategy that promotes Ca uptake into mitochondria might be a new and safer option for treating VAs in HF. Funding Acknowledgement Type of funding source: None

Inhibition of Autophagy in Cardiomyocytes by Calmodulin Antagonists

Macroautophagy (hereafter referred to as autophagy) is a catabolic process that regulates important aspects of myocardial metabolism. While numerous studies have indicated involvement of signaling via Ca2+ in autophagic flux, the precise mechanisms remain unknown. Calmodulin (CaM) is a ubiquitous transducer of intracellular Ca2+ signals by interacting with an estimated network of 300 cellular proteins. We have begun to examine the role of CaM in autophagic flux in cardiomyocytes. A basal level of autophagy is required for normal myocardial function. Chloroquine dose‐dependently reduces basal autophagic flux in H9c2 cardiomyocytes, as indicated by LC3 immunoblotting, p62 immunofluorescence, and autophagic flux assays. Competitive binding assays using purified CaM and FRET‐based CaM biosensors demonstrate direct Ca2+‐dependent CaM binding to chloroquine. The structurally distinct CaM antagonists W‐7, trifluoperazine, and CGS 9343B also dose‐dependently inhibit basal autophagic flux by reducing autophagosome degradation. Most CaM‐dependent processes depend at the front end on the amplitude and dynamics of intracellular Ca2+ signals. Interestingly, chloroquine, W‐7, trifluoperazine, and CGS 9343b all mobilize intracellular Ca2+. The effects of these pharmacological agents to inhibit autophagic flux are thus associated with their inhibition of CaM and not of initial Ca2+ signals produced in cells. These data indicate a role for CaM in the control of autophagic flux in cardiomyocytes.Support or Funding InformationAmerican Heart Association (15SDG25090279) and Iowa Osteopathic Education and Research Funds (Eric Wauson); NIH HL112184 and DMU IOER‐090717 (Quang‐Kim Tran)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Size Matters: Ryanodine Receptor Cluster Size Heterogeneity Potentiates Calcium Waves

Ryanodine receptors (RyRs) mediate calcium (Ca)-induced Ca release and intracellular Ca homeostasis. In a cardiac myocyte, RyRs group into clusters of variable size from a few to several hundred RyRs, creating a spatially nonuniform intracellular distribution. It is unclear how heterogeneity of RyR cluster size alters spontaneous sarcoplasmic reticulum (SR) Ca releases (Ca sparks) and arrhythmogenic Ca waves. Here, we tested the impact of heterogeneous RyR cluster size on the initiation of Ca waves. Experimentally, we measured RyR cluster sizes at Ca spark sites in rat ventricular myocytes and further tested functional impacts using a physiologically detailed computational model with spatial and stochastic intracellular Ca dynamics. We found that the spark frequency and amplitude increase nonlinearly with the size of RyR clusters. Larger RyR clusters have lower SR Ca release threshold for local Ca spark initiation and exhibit steeper SR Ca release versus SR Ca load relationship. However, larger RyR clusters tend to lower SR Ca load because of the higher Ca leak rate. Conversely, smaller clusters have a higher threshold and a lower leak, which tends to increase SR Ca load. At the myocyte level, homogeneously large or small RyR clusters limit Ca waves (because of low load for large clusters but low excitability for small clusters). Mixtures of large and small RyR clusters potentiates Ca waves because the enhanced SR Ca load driven by smaller clusters enables Ca wave initiation and propagation from larger RyR clusters. Our study suggests that a spatially heterogeneous distribution of RyR cluster size under pathological conditions may potentiate Ca waves and thus afterdepolarizations and triggered arrhythmias.

The effects of Tl+ ions on the dynamics of intracellular Ca2+ in rat cardiomyocytes

The effects of Т1+ ions on the dynamics of intracellular Cа2+ in neonatal rat cardiomyocytes have been studied. It was shown for the first time that application of Т1+ led to an uncontrolled increase in [Cа2+]i in cells. Moreover, the ability of Т1+ to increase [Cа2+]i depended on the Т1+ concentration used and the time of exposure to the cells. The increase in [Cа2+]i was related to the entry of Cа2+ from the extracellular medium. Thallium did not release Cа2+ from intracellular stores. The thallium-induced increase in [Cа2+]i was not inhibited by nifedipine. It is possible that L-channels do not participate in the processes of thalliuminduced increase in [Cа2+]i. It is assumed that the thallium ions-induced calcium overload in cardiomyocytes may contribute to the toxic effect of Т1+ on the myocardium.

Changing calcium: CRAC channel (STIM and Orai) expression, splicing, and posttranslational modifiers.

A wide variety of cellular function depends on the dynamics of intracellular Ca(2+) signals. Especially for relatively slow and lasting processes such as gene expression, cell proliferation, and often migration, cells rely on the store-operated Ca(2+) entry (SOCE) pathway, which is particularly prominent in immune cells. SOCE is initiated by the sensor proteins (STIM1, STIM2) located within the endoplasmic reticulum (ER) registering the Ca(2+) concentration within the ER, and upon its depletion, cluster and trap Orai (Orai1-3) proteins located in the plasma membrane (PM) into ER-PM junctions. These regions become sites of highly selective Ca(2+) entry predominantly through Orai1-assembled channels, which, among other effector functions, is necessary for triggering NFAT translocation into the nucleus. What is less clear is how the spatial and temporal spread of intracellular Ca(2+) is shaped and regulated by differential expression of the individual SOCE genes and their splice variants, their heteromeric combinations and pre- and posttranslational modifications. This review focuses on principle mechanisms regulating expression, splicing, and targeting of Ca(2+) release-activated Ca(2+) (CRAC) channels.

A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels.

STUDY QUESTIONCould drugs targeting ATP-sensitive K+ (KATP) channels prevent any spontaneous increase in intracellular Ca2+ that may occur in human metaphase II (MII) oocytes under in vitro conditions?SUMMARY ANSWERPinacidil, a KATP channel opener, and glibenclamide, a KATP channel blocker, prevent a spontaneous increase in intracellular Ca2+ in human MII oocytes.WHAT IS KNOWN ALREADYThe quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca2+ homeostasis is crucial for cell wellbeing and increased intracellular Ca2+ levels is a well-established indicator of cell stress.STUDY DESIGN, SIZE, DURATIONSupernumerary human oocytes (n = 102) collected during IVF/ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca2+ levels were monitored in oocytes in the following experimental groups: (i) Control, (ii) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP.PARTICIPANTS/MATERIALS/SETTINGS/METHODSOocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean ± SD) age 34.1 ± 0.6 years, n = 41. Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca2+-sensitive dye, and monitored by laser confocal microscopy for 2 h at 10 min intervals. Time between oocyte collection and start of Ca2+ monitoring was 80.4 ± 2.1 h.MAIN RESULTS AND THE ROLE OF CHANCEIntracellular levels of Ca2+ increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0 ± 11.8 AU (AU = arbitrary units; n = 23) to 91.8 ± 14.0 AU (n = 19; P < 0.001) after 2 h of monitoring. Pinacidil (100 µM) inhibited this increase in Ca2+ (85.3 ± 12.3 AU at the beginning of the experiment, 81.7 ± 11.0 AU at the end of the experiment; n = 13; P = 0.616). Glibenclamide (100 µM) also inhibited the increase in Ca2+ (74.7 ± 10.6 AU at the beginning and 71.8 ± 10.9 AU at the end of the experiment; n = 13; P = 0.851. DNP (100 mM) induced an increase in intracellular Ca2+ that was inhibited by glibenclamide (100 µM; n = 9) but not by pinacidil (100 µM; n = 5).LIMITATIONS, REASONS FOR CAUTIONOwing to clinical and ethical considerations, it was not possible to monitor Ca2+ in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4 ± 2.1 h (n = 102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilized, it is possible that they may have been abnormal with impaired Ca2+ homeostasis and, furthermore, the altered Ca2+ homeostasis might have been associated solely with the protracted incubation.WIDER IMPLICATIONS OF THE FINDINGSThese results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca2+, which can be counteracted by drugs targeting KATP channels. As Ca2+ homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that KATP channel openers and blockers should be tested as drugs for improving success rates of ART.STUDY FUNDING/COMPETING INTEREST(S)University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest.

PCB-95 Modulates the Calcium-Dependent Signaling Pathway Responsible for Activity-Dependent Dendritic Growth

Background: Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) promote dendritic growth in hippocampal neurons via ryanodine receptor (RyR)-dependent mechanisms; however, downstream signaling events that link enhanced RyR activity to dendritic growth are unknown. Activity-dependent dendritic growth, which is a critical determinant of neuronal connectivity in the developing brain, is mediated by calcium ion (Ca2+)-dependent activation of Ca2+/calmodulin kinase-I (CaMKI), which triggers cAMP response element binding protein (CREB)-dependent Wnt2 transcription. RyRs regulate the spatiotemporal dynamics of intracellular Ca2+ signals, but whether RyRs promote dendritic growth via modulation of this signaling pathway is not known.Objective: We tested the hypothesis that the CaMKI–CREB–Wnt2 signaling pathway couples NDL PCB-enhanced RyR activity to dendritic arborization.Methods and Results: Ca2+ imaging of dissociated cultures of primary rat hippocampal neurons indicated that PCB-95 (2,2´,3,5´6-pentachlorobiphenyl; a potent RyR potentiator), enhanced synchronized Ca2+ oscillations in somata and dendrites that were blocked by ryanodine. As determined by Western blotting and quantitative polymerase chain reaction, PCB-95 also activated CREB and up-regulated Wnt2. Blocking CaMKK, CaMKIα/γ, MEK/ERK, CREB, or Wnt2 prevented PCB-95–induced dendritic growth. Antagonism of γ-aminobutyric acid (GABA) receptors with bicuculline (BIC) phenocopied the dendrite-promoting effects of PCB-95, and pharmacological antagonism or siRNA knockdown of RyR blocked BIC-induced dendritic growth in dissociated and slice cultures of hippocampal neurons.Conclusions: RyR activity contributes to dynamic remodeling of dendritic architecture in response to NDL PCBs via CaMKI–CREB–Wnt2 signaling in rats. Our findings identify PCBs as candidate environmental risk factors for neurodevelopmental disorders, especially in children with heritable deficits in calcium signaling associated with autism.

PCB-95 Promotes Dendritic Growth via Ryanodine Receptor–Dependent Mechanisms

Background: Aroclor 1254 (A1254) interferes with normal dendritic growth and plasticity in the developing rodent brain, but the mechanism(s) mediating this effect have yet to be established. Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) enhance the activity of ryanodine receptor (RyR) calcium ion (Ca2+) channels, which play a central role in regulating the spatiotemporal dynamics of intracellular Ca2+ signaling. Ca2+ signaling is a predominant factor in shaping dendritic arbors, but whether PCB potentiation of RyR activity influences dendritic growth is not known.Objective: We determined whether RyR activity is required for PCB effects on dendritic growth.Methods and Results: Golgi analysis of hippocampi from weanling rats confirmed that developmental exposure via the maternal diet to NDL PCB-95 (2,2´,3,5´6-pentachlorobiphenyl), a potent RyR potentiator, phenocopies the dendrite-promoting effects of A1254. Dendritic growth in dissociated cultures of primary hippocampal neurons and in hippocampal slice cultures is similarly enhanced by PCB-95 but not by PCB-66 (2,3,4´,4-tetrachlorobiphenyl), a congener with negligible effects on RyR activity. The dendrite-promoting effects of PCB-95 are evident at concentrations as low as 2 pM and are inhibited by either pharmacologic blockade or siRNA knockdown of RyRs.Conclusions: Our findings demonstrate that environmentally relevant levels of NDL PCBs modulate neuronal connectivity via RyR-dependent effects on dendritic arborization. In addition, these findings identify RyR channel dysregulation as a novel mechanism contributing to dysmorphic dendritogenesis associated with heritable and environmentally triggered neurodevelopmental disorders.

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

Ca2+ Regulation of Mitochondrial ATP Synthesis Visualized at the Single Cell Level

Intracellular Ca(2+) levels play a crucial role in the control of ATP synthesis. However, the spatiotemporal correlation between ATP and Ca(2+) remains unclear due to the inability to visualize these factors within same individual cells. A Förster resonance energy transfer (FRET)-based fluorescent ATP probe, named ATeam, was recently developed for ATP imaging in single living cells. However, the spectra of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) used as the FRET donor and the acceptor, respectively, significantly overlap with the ultraviolet-excitable Ca(2+) probe, fura-2. In the present work, we developed new red-shifted ATP probes, GO-ATeams, in which green fluorescent protein (GFP) and orange fluorescent protein (OFP) was used as the FRET pair to minimize spectral overlap with the fura-2 emission. The dynamics of intracellular Ca(2+) and mitochondrial ATP levels in single histamine-stimulated HeLa cells were successfully visualized by using fura-2 and GO-ATeam. The experiments showed that histamine induced increases of both intracellular Ca(2+) and mitochondrial ATP levels. The increment of mitochondrial ATP levels was proportional to that of Ca(2+). This finding suggests that cellular Ca(2+) levels might precisely control mitochondrial ATP synthesis in response to the increased ATP consumption triggered by Ca(2+). In addition, GO-ATeam has several advantages over the original ATeam. The GO-ATeam signal was more stable against acidification, which would allow ATP imaging inside acidic intracellular compartments. Also, the GO-ATeam excitation wavelength is much less phototoxic to cells, making the probe suitable for long-time observation.

Mechanisms of heterogeneity of calcium response to kainate and neuronal types in rat cortical primary culture

The dynamics of intracellular Ca2+ signal in response to NMDA (N-methyl-D-aspartate, 30 μM) or KA (kainite, 30 μM), its dependence on extracellular Ca2+ and the mechanisms of KA-triggered Ca2+ entry into neurons have been tested in neurons of rat cortical primary cultures. The level of intracellular free Ca2+ concentrations ([Ca2+]i) was evaluated on Leica SP5 MF confocal microscope using Fluo-3 fluorescent dye, which resolves changes in [Ca2+]i in the micromolar range. The dynamics of [Ca2+]i increase in response to NMDA and KA was different but in both cases the [Ca2+]i increase required the presence of Ca2+ in the extracellular solution. The neuronal population was found to be heterogeneous, based on the response to KA applied together with either L-type calcium channel blocker nifedipine (3 μM) or IEM-1460 (3 μM), a blocker of Ca2+-permeable AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) lacking GluR2 subunit. Experiments exhibited three types of calcium responses, characteristically belonging to interneurons (expressing Ca2+-permeable AMPAR), pyramidal neurons (with AMPAR containing GluR2, making them impermeable to Ca2+), and intermediate type of cells expressing both AMPAR types. Thus, we have demonstrated the role of AMPAR and L-type calcium channels in KA-triggered Ca2+ entry into neurons. The dynamics of [Ca2+]i during the KA treatment was shown to depend on subunit composition of particular AMPAR subtype expressed in neurons. The data suggest that neuronal types existing in adult cortical tissue are probably presented in primary culture, too.

Integrative modeling of the cardiac ventricular myocyte

Cardiac electrophysiology is a discipline with a rich 50-year history of experimental research coupled with integrative modeling which has enabled us to achieve a quantitative understanding of the relationships between molecular function and the integrated behavior of the cardiac myocyte in health and disease. In this paper, we review the development of integrative computational models of the cardiac myocyte. We begin with a historical overview of key cardiac cell models that helped shape the field. We then narrow our focus to models of the cardiac ventricular myocyte and describe these models in the context of their subcellular functional systems including dynamic models of voltage-gated ion channels, mitochondrial energy production, ATP-dependent and electrogenic membrane transporters, intracellular Ca dynamics, mechanical contraction, and regulatory signal transduction pathways. We describe key advances and limitations of the models as well as point to new directions for future modeling research. WIREs Syst Biol Med 2011 3 392-413 DOI: 10.1002/wsbm.122

Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2+ waves in interstitial cells of Cajal‐like cells of the rabbit urethra

Although spontaneous Ca(2+) waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca(2+) release, the interactions among mitochondrial Ca(2+) buffering, cellular energetics and ER Ca(2+) release in determining the spatiotemporal dynamics of intracellular Ca(2+) remain to be elucidated. Spontaneous Ca(2+) transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca(2+) imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red. Spontaneous Ca(2+) waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca(2+) dynamics were characterized by a gradual rise in basal Ca(2+) that preceded each regenerative Ca(2+) transient. Caffeine evoked oscillatory Ca(2+) waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca(2+) wave generation with a rise in basal Ca(2+), and subsequent caffeine evoked a single rudimentary Ca(2+) transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca(2+) and abolished Ca(2+) waves. However, caffeine still induced oscillatory Ca(2+) transients. Mitochondrial Ca(2+) uptake inhibition with RU360 attenuated Ca(2+) wave amplitudes, while mitochondrial Ca(2+) efflux inhibition with CGP37157 suppressed the initial Ca(2+) rise to reduce Ca(2+) wave frequency. Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca(2+) wave generation and may regulate ER Ca(2+) release frequency by buffering Ca(2+) within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca(2+) buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.

A Simulation Study of Calcium Dynamics Features Caused by Exchange between the Cytosol and Organellar Stores of Neurons

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron. The fragments contained the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER (model 2), functioning as calcium stores. With simulating single excitatory synaptic actions, we examined the dependence of the dynamics of intracellular Ca 2+ levels on the maximum rate of Ca 2+ exchange between the cytosol and these stores, as well as on the intensity of the diffusion flow into adjacent organelle-free regions. The plasma membrane of the compartment had ion channels (including those of the synaptic current) and the calcium pump characteristic of the mentioned neurons. The model equations took into account Ca 2+ exchange between the cytosol, extracellular environment, and organellar stores, as well as the diffusion process. In model 1, the mitochondria exchanged Ca 2+ with the cytosol through the uniporter and sodium-calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also included. In model 2, the ER membrane had the calcium pump, leak channels, and channels of calcium-induced and inositol-3-phosphate-dependent Ca 2+ release. The stores (mitochondria or ER) occupied 36% of the total volume of the compartment. An increase in the maximum rate of calcium exchange with the stores led to a proportional decrease in the peak Ca 2+ concentrations in the cytosol ([Ca 2+ ] i ), more pronounced in the case of the ER; the Ca 2+ concentration in both types of stores increased significantly. Due to the higher storage rate, the ER was able to absorb several times greater amounts of Ca 2+ than the mitochondria did. With smaller diffusion flux (e.g., similarly to the case of diffusion from a larger-sized head into the neck of the dendritic spine), the intensity of cytosolic transients increased at fixed kinetics of flux exchange with the stores. Therefore, the organellar stores can significantly modulate not only the intensity but also the time course of changes in the intracellular Ca 2+ levels.

Inositol trisphosphate receptor and ion channel models based on single-channel data

The inositol trisphosphate receptor (IPR) plays an important role in controlling the dynamics of intracellular Ca(2+). Single-channel patch-clamp recordings are a typical way to study these receptors as well as other ion channels. Methods for analyzing and using this type of data have been developed to fit Markov models of the receptor. The usual method of parameter fitting is based on maximum-likelihood techniques. However, Bayesian inference and Markov chain Monte Carlo techniques are becoming more popular. We describe the application of the Bayesian methods to real experimental single-channel data in three ion channels: the ryanodine receptor, the K(+) channel, and the IPR. One of the main aims of all three studies was that of model selection with different approaches taken. We also discuss the modeling implications for single-channel data that display different levels of channel activity within one recording.