Ca Oscillations Research Articles

Dissecting the roles of calcium cycling and its coupling with voltage in the genesis of early afterdepolarizations in cardiac myocyte models.

Early afterdepolarizations (EADs) are abnormal depolarizations during the plateau phase of the action potential, which are known to be associated with lethal arrhythmias in the heart. There are two major hypotheses for EAD genesis based on experimental observations, i.e., the voltage (Vm)-driven and intracellular calcium (Ca)-driven mechanisms. In ventricular myocytes, Ca and Vm are bidirectionally coupled, which can affect each other's dynamics and result in new dynamics, however, the roles of Ca cycling and its coupling with Vm in the genesis of EADs have not been well understood. In this study, we use an action potential model that is capable of independent Vm and Ca oscillations to investigate the roles of Vm and Ca coupling in EAD genesis. Four different mechanisms of EADs are identified, which are either driven by Vm oscillations or Ca oscillations alone, or oscillations caused by their interactions. We also use 5 other ventricular action potential models to assess these EAD mechanisms and show that EADs in these models are mainly Vm-driven. These mechanistic insights from our simulations provide a theoretical base for understanding experimentally observed EADs and EAD-related arrhythmogenesis.

Synaptic plasticity via receptor tyrosine kinase/G-protein-coupled receptor crosstalk

Cellular signaling involves a large repertoire of membrane receptors operating in overlapping spatiotemporal regimes and targeting many common intracellular effectors. However, both the molecular mechanisms and the physiological roles of crosstalk between receptors, especially those from different superfamilies, are poorly understood. We find that the receptor tyrosine kinase (RTK) TrkB and the G-protein-coupled receptor (GPCR) metabotropic glutamate receptor 5 (mGluR5) together mediate hippocampal synaptic plasticity in response to brain-derived neurotrophic factor (BDNF). Activated TrkB enhances constitutive mGluR5 activity to initiate a mode switch that drives BDNF-dependent sustained, oscillatory Ca2+ signaling and enhanced MAP kinase activation. This crosstalk is mediated, in part, by synergy between Gβγ, released by TrkB, and Gαq-GTP, released by mGluR5, to enable physiologically relevant RTK/GPCR crosstalk.

Both electrical and metabolic coupling shape the collective multimodal activity and functional connectivity patterns in beta cell collectives: A computational model perspective.

Pancreatic beta cells are coupled excitable oscillators that synchronize their activity via different communication pathways. Their oscillatory activity manifests itself on multiple timescales and consists of bursting electrical activity, subsequent oscillations in the intracellular Ca^{2+}, as well as oscillations in metabolism and exocytosis. The coordination of the intricate activity on the multicellular level plays a key role in the regulation of physiological pulsatile insulin secretion and is incompletely understood. In this paper, we investigate theoretically the principles that give rise to the synchronized activity of beta cell populations by building up a phenomenological multicellular model that incorporates the basic features of beta cell dynamics. Specifically, the model is composed of coupled slow and fast oscillatory units that reflect metabolic processes and electrical activity, respectively. Using a realistic description of the intercellular interactions, we study how the combination of electrical and metabolic coupling generates collective rhythmicity and shapes functional beta cell networks. It turns out that while electrical coupling solely can synchronize the responses, the addition of metabolic interactions further enhances coordination, the spatial range of interactions increases the number of connections in the functional beta cell networks, and ensures a better consistency with experimental findings. Moreover, our computational results provide additional insights into the relationship between beta cell heterogeneity, their activity profiles, and functional connectivity, supplementing thereby recent experimental results on endocrine networks.

Human‐Derived Induced Pluripotent Stem Cells‐Based Disease Models of the Alzheimer’s Disease (AD) Patients Expressed AD Phenotypes and Responded to 17β‐Estradiol Treatment

AbstractBackgroundNeuronal models of both familial and sporadic cases of Alzheimer’s Disease (fAD/sAD) were generated from the human‐derived induced pluripotent stem cells (iPSCs) of AD patients. Using this in vitro cellular models, AD‐like phenotypes was analyzed with or without 17β‐estradiol, the sex hormone that are supposed to be involved in the AD pathogenesis.Method5 lines of iPSCs comprised of 1 healthy control, 2 fAD, 2 sAD were differentiated to neurons with a newly established neuronal differentiation method. The AD‐like phenotypes such as secreted Aβ peptides and neuronal excitability were measured by ELISA and Ca‐imaging (Fluo‐8 indicator). In addition, the neuronal complexity and excitability were examined after long (4 days) and short (15 minutes) 17β‐estradiol treatment, respectively.ResultThe neurons were successfully induced from the iPSCs with the newly established differentiation method. The neurons induced from AD lines secreted more Aβ(1‐40) and Aβ(1‐42) peptides and increased Ca oscillations compared to the control line. The treatment with 17β‐estradiol to neurons promoted the neuronal complexity and cellular excitability.ConclusionNeurons derived from the iPSCs of the AD patients expressed AD phenotypes and responded to the treatment of 17β‐estradiol. Thus, this suggested the therapeutic effects of sex steroid hormones to AD can be evaluated in human cells. This enables us to examine the variability of estradiol response among patients and plan clinical trials based on the responsiveness.

Photopharmacological dissociation of transcriptional activation and degranulation of mast cells.

Besides a key role in allergic diseases, mast cells confer important modulatory functions in innate and adaptive immunity. The communication between mast cells and other immune cells is not only based on the acute or long-term release of pre-stored mediators involving distinct processes of degranulation but also on the expression of specific surface proteins. Activation of mast cells typically involves Ca2+ signalling, which evokes both transcriptional activation and degranulation. To dissect the complex functions of mast cells in human pathology and to explore new therapeutic strategies we aimed towards specific manipulation of mast cell phenotype. Characterization of an optochemogenetic protocol for activation RBL-2H3 mast cells revealed that repetitive photocycling of OptoBI-1 to generate oscillatory Ca2+ signals elicits rapid and efficient NFAT nuclear translocation without concomitant degranulation. By contrast, conventional pharmacological activation of overexpressed TRPC6 channel was strictly associated with CD63-associated secretion. Our results suggest that the specific modulation of mast cell phenotype as obtained by TRPC6-OptoBI-1-mediated activation, arises from introduction of a highly specific conformational and functional state of the TRPC6 channel, resulting in unique Ca2+signatures. Further understanding of these phenomena may lead to new approaches for (photo)pharmacological immunomodulation.

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.

Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X.

Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2-3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

Cholesterol-induced robust Ca oscillation in astrocytes required for survival and lipid droplet formation in high-cholesterol condition

SummaryCholesterol, one of the major cell membrane components, stabilizes membrane fluidity and regulates signal transduction. Beside its canonical roles, cholesterol has been reported to directly activate signaling pathways such as hedgehog (Hh). We recently found that astrocytes, one of the glial cells, respond to Hh pathway stimulation by Ca signaling. These notions led us to test if extracellularly applied cholesterol triggers Ca signaling in astrocytes. Here, we found that cholesterol application induces robust Ca oscillation only in astrocytes with different properties from the Hh-induced Ca response. The Ca oscillation has a long delay which corresponds to the onset of cholesterol accumulation in the plasma membrane. Blockade of the Ca oscillation resulted in enhancement of astrocytic cell death and disturbance of lipid droplet formation, implying a possibility that the cholesterol-induced Ca oscillation plays important roles in astrocytic survival and cholesterol handling under pathological conditions of cholesterol load such as demyelination.

Constitutive activation of a nuclear-localized calcium channel complex in Medicago truncatula

Nuclear Ca2+ oscillations allow symbiosis signaling, facilitating plant recognition of beneficial microsymbionts, nitrogen-fixing rhizobia, and nutrient-capturing arbuscular mycorrhizal fungi. Two classes of channels, DMI1 and CNGC15, in a complex on the nuclear membrane, coordinate symbiotic Ca2+ oscillations. However, the mechanism of Ca2+ signature generation is unknown. Here, we demonstrate spontaneous activation of this channel complex, through gain-of-function mutations in DMI1, leading to spontaneous nuclear Ca2+ oscillations and spontaneous nodulation, in a CNGC15-dependent manner. The mutations destabilize a hydrogen-bond or salt-bridge network between two RCK domains, with the resultant structural changes, alongside DMI1 cation permeability, activating the channel complex. This channel complex was reconstituted in human HEK293T cell lines, with the resultant calcium influx enhanced by autoactivated DMI1 and CNGC15s. Our results demonstrate the mode of activation of this nuclear channel complex, show that DMI1 and CNGC15 are sufficient to create oscillatory Ca2+ signals, and provide insights into its native mode of induction.

Substrate stiffness-dependent regulatory volume decrease and calcium signaling in chondrocytes.

The pericellular matrix stiffness is strongly associated with its biochemical and structural changes during the aging and osteoarthritis progress of articular cartilage. However, how substrate stiffness modulates the chondrocyte regulatory volume decrease (RVD) and calcium signaling in chondrocytes remains unknown. This study aims to investigate the effects of substrate stiffness on the chondrocyte RVD and calcium signaling by recapitulating the physiologically relevant substrate stiffness. Our results showed that substrate stiffness induces completely different dynamical deformations between the cell swelling and recovering progresses. Chondrocytes swell faster on the soft substrate but recovers slower than the stiff substrate during the RVD response induced by the hypo-osmotic challenge. We found that stiff substrate enhances the cytosolic Ca oscillation of chondrocytes in the iso-osmotic medium. Furthermore, chondrocytes exhibit a distinctive cytosolic Ca oscillation during the RVD response. Soft substrate significantly improves the Ca oscillation in the cell swelling process whereas stiff substrate enhances the cytosolic Ca oscillation in the cell recovering process. Our work also suggests that the TRPV4 channel is involved in the chondrocyte sensing substrate stiffness by mediating Ca signaling in a stiffness-dependent manner. This helps to understand a previously unidentified relationship between substrate stiffness and RVD response under the hypo-osmotic challenge. A better understanding of substrate stiffness regulating chondrocyte volume and calcium signaling will aid the development of novel cell-instructive biomaterial to restore cellular functions.

SARS-CoV-2 deregulates the vascular and immune functions of brain pericytes via Spike protein

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Glucose-induced [Ca2+]i oscillations in β cells are composed of trains of spikes within a subplasmalemmal microdomain

Electrical activity and oscillations of cytosolic Ca2+ concentrations ([Ca2+]i) that trigger insulin release in response to glucose are key functions of pancreatic β cells. Although oscillatory Ca2+ signals have been intensively studied in β cells, their lower frequency did not match that of electrical activity. In addition, the measured peak [Ca2+]i did not reach levels that are typically required by synaptotagmins to elicit the release of insulin-containing vesicles in live-cell experiments. We therefore sought to resolve the Ca2+ dynamics in the subplasmalemmal microdomain that is critical for triggering fast exocytosis. Applying total internal reflection fluorescence (TIRF) microscopy in insulin-producing INS-1E and primary mouse β cells, we resolved extraordinary fast trains of Ca2+ spiking (frequency > 3 s−1) in response to glucose exposure. Using a low-affinity [Ca2+]i indicator dye, we provide experimental evidence that Ca2+ spikes reach low micromolar apparent concentrations in the vicinity of the plasma membrane. Analysis of Ca2+ spikes evoked by repeated depolarization for 10 ms closely matched the Ca2+ dynamics observed upon glucose application. To our knowledge, this is the first study that experimentally demonstrates Ca2+ spikes in β cells with velocities that resemble those of bursting or continuously appearing trains of action potentials (APs) in non-patched cells.

Ca2+ Oscillations and NFATC1 -Activation Enable Oncogenic Signaling in Pre-B Cell Transformation and Leukemogenesis

Background: Nuclear factor of activated T cells (NFATC1) regulates activation and differentiation of B and T cells. Activation of NFATC1 requires sustained Ca2+ influx by store-operated Ca2+ entry (SOCE). Our group previously showed that oncogenic signaling from the BCR-ABL1 kinase in Ph+ ALL results in autonomous oscillatory Ca2+ signaling (Klein et al., J Exp Med 2004; Feldhahn et al., J Exp Med 2005). However, the significance of Ca2+ oscillations remained unclear. Here we examined the function of Orai and Stim1/2, the central mediators of SOCE, and the role of Ca2+ oscillations in the survival of BCR-ABL1 -driven pre-B ALL cells.Results: To investigate the consequence of inducible deletion of SOCE-effector genes, Orai1, Stim1, and Stim2, we have generated BCR-ABL1 - and CreERT2-transduced murine pre-B Orai1f/f and Stim1/2f/f cells. Both Orai1 - and Stim1/2 -deletion impaired Ca2+ influx from the extracellular space when Ca2+ uptake from cytosol to endoplasmic reticulum was pharmacologically inhibited. Consistently, both Orai1 - and Stim1/2 -deletion significantly attenuated the amplitude and frequency of autonomous Ca2+ oscillations, compared with that of empty vector controls. To determine whether the impaired Ca2+ signaling associated with cell survival and proliferation, we next analyzed their growth and colony-forming capacity. As expected, both Orai1 - and Stim1/2 -deleted pre-B ALL cells exhibited growth disadvantage and reduced colony-forming capacity, compared with empty vector controls. Flow cytometry studies of Annexin V revealed that both Orai1 - and Stim1/2 -deletion induced apoptosis in the BCR-ABL1 -transformed cells. These results suggest that autonomous Ca2+ oscillations are needed to circumvent apoptosis, and enhance cell growth and self-renewal in BCR-ABL-driven ALL cells. Interestingly, even in normal pre-B cells and in the absence of autonomous Ca2+ oscillations, both Orai1 - and Stim1/2 -deletion caused growth disadvantage suggesting the existence of similar cellular function to Ca2+ oscillations in normal pre-B cells. Subsequently, we studied NFATc1 activity by Western blot, as reflected by nuclear localization, in pre-B ALL cells before and after Orai1 - and Stim1/2 -deletion. NFATc1 is known as one of a central NFAT family member that is activated in lymphocytes upon BCR and TCR stimulation. While Orai1fl/fl and Stim1/2fl/fl B-lineage ALL cells transduced with empty vector controls displayed marked nuclear accumulation of NFATc1, Cre-mediated deletion of Orai1 and Stim1/2 strongly reduced nuclear accumulation of NFATc1, with slight increases in the cytoplasmic fraction. To examine the consequences of NFATc1 inhibition, we generated BCR-ABL1 - and CreERT2-transduced murine pre-B Nfatc1f/f and observed its effect on cell growth. As expected, inducible ablation of Nfatc1 resulted in rapid depletion of pre-B ALL cells from the cell culture compared to empty vector controls. Importantly, a NFAT-calcineurin association inhibitor, INCA-6, displayed growth inhibitory effects in all six patient-derived xenograft of B cell ALL tested, including samples from patients who relapsed after initially successful chemotherapy. These results suggest that the Ca2+-NFAT signal may suppress apoptosis, and promote drug-resistance and relapse in human pre-ALL cells.Conclusions: Taken together, our findings showed that SOCE has a critical role in NFAT activation and survival of BCR-ABL1 -driven pre-B ALL cells. Orai1- and Stim1/2-mediated autonomous Ca2+ oscillatory signal is required for BCR-ABL1-dependent proliferation. This signal may bypass the NFAT activation which suppress apoptosis and enhance proliferation in BCR-ABL-driven ALL cells. DisclosuresNo relevant conflicts of interest to declare.

123-OR: Deletion of Both Pcsk1 And Pcsk2 in Beta Cells: Lack of Mature Insulin Drives Beta-Cell Expansion and Dysfunction, but Not Severe Diabetes in Mice

Proinsulin (PI) is processed to mature insulin by prohormone convertases 1/3 (Pcsk1) and 2 (Pcsk2). In individuals with T1D and T2D, there is an increase in the circulating PI:insulin ratio. We hypothesized that β-cell Pcsk1 and Pcsk2 deletion would result in β-cell dysfunction, loss of insulin production, and marked hyperglycemia. Single Pcsk1flox/flox Ins1cre/+ (Pcsk1βKO), Pcsk2flox/flox Ins1cre/+ (Pcsk2βKO), and double Pcsk1flox/flox Pcsk2flox/flox Ins1cre/+ (DPCβKO) β-cell prohormone convertase knockout mice were generated (with respective Pcsk1+/+ Pcsk2+/+ Ins1cre/+ controls) and monitored until 30 weeks old. Pcsk2βKO mice displayed no glycemic phenotype, while Pcsk1βKO mice displayed mild hyperglycemia and impaired glucose tolerance by 26 weeks of age. DPCβKO mice displayed mild hyperglycemia (10.1±1.6 vs. 12.3±2.1 mM; p=0.006) and marked glucose intolerance by 10 weeks of age (AUC: 418±135 vs. 1485±360; p<0.0001). No animals displayed obesity relative to controls. Insulin+ pancreas area was doubled in DPCβKO male mice (p=0.0004) and DPCβKO islets showed early glucose-induced Ca2+ influx and loss of oscillatory Ca2+ flux. Pcsk2βKO islets had unaltered mature insulin, while Pcsk1βKO islets had reduced mature insulin. DPCβKO islets contained no detectable mature insulin by western blot. In contrast, Pcsk2βKO islets had markedly reduced mature islet amyloid polypeptide (IAPP), while Pcsk1βKO islets contained normal mature IAPP levels. Our results suggest that complete loss of β-cell prohormone processing, including PI processing, can drive β-cell dysfunction and expansion, but only mild diabetes and glucose intolerance. Although Pcsk2 deletion in β cells does not impact glycemia or PI processing, it is required for normal proIAPP processing. In total, our data support the idea that altered β-cell prohormone processing does not impact obesity but can contribute to the pathogenesis of diabetes via β-cell dysfunction. Disclosure A. Taylor: None. Y. Chen: None. D. Pasula: None. D. S. Luciani: None. B. Verchere: Advisory Panel; Self; Integrated Nanotherapeutics Inc., Sirona Biochem, Stock/Shareholder; Self; Integrated Nanotherapeutics Inc. Funding Canadian Institutes of Health Research

On the study of seasonal patterns of 25–70-day intraseasonal oscillations in Central Africa

With its strong influence on weather and climate, the Madden–Julian Oscillation (MJO) plays a crucial role in the tropical climate system. However, one of the greatest challenges of climate modeling remains definition of suitable MJO-related metrics for the efficient use in climate models. This requires a good documentation of the MJO variation patterns throughout the year and from tear to another. As contribution to the definition of these MJO-related metrics in General Circulation Models (GCMs), this paper addresses the seasonal patterns of 25–70-day Intraseasonal Oscillations (ISO) in CA using the 2.5° × 2.5° daily Outgoing Longwave Radiation (OLR), for the period 1981–2015 (35 years). One novelty in this study is the use of a Discrete Wavelet Transform (DWT) model to compute ISO amplitude and frequency indices and then investigate their variations from one season to another. The analyses showed that the ISO characteristics (amplitude, frequency and propagation speed) strongly vary from season to another. The ISO intensity (ISOI) is extremely high during December–February (DJF) and March–May (MAM) and lower during June–August (JJA) and September–November (SON) seasons. As for the ISO period (ISOP), the low-frequency ISO (high ISOP) are observed during DJF and JJA and the high frequency (low ISOP) during MAM and SON seasons. However, for the four seasons, the period is predominantly contained between 40 and 50 days, suggesting the dominance of MJO. The investigation of propagation characteristics for the selected Active Intraseasonal Events (AIEs) found the eastward propagation to be the dominant feature in all seasons but the propagation speed is lower for DJF and MAM, when compared with JJA and SON. At global scale, we found that the incursions of the moisture fluxes from Atlantic and Indian oceans are responsible for the convection and subsequently the rainfall enhancement over CA at intraseasonal timescales. We also accessed the impact of ISO on rainfall using and index called the Impact Rate (IR). The plots of spatial distribution of IR during the four seasons showed that the influence of ISO on rainfall varies from one season to another, with some zones of very high IR and other zones of lower IR. Moreover, the zone of high IR propagates northwards from one season to another.

Modeling of Ca2+ transients initiated by GPCR agonists in mesenchymal stromal cells

The integrative study that included experimentation and mathematical modeling was carried out to analyze dynamic aspects of transient Ca2+ signaling induced by brief pulses of GPCR agonists in mesenchymal stromal cells from the human adipose tissue (AD-MSCs). The experimental findings argued for IP3/Ca2+-regulated Ca2+ release via IP3 receptors (IP3Rs) as a key mechanism mediating agonist-dependent Ca2+ transients. The consistent signaling circuit was proposed to formalize coupling of agonist binding to Ca2+ mobilization for mathematical modeling. The model properly simulated the basic phenomenology of agonist transduction in AD-MSCs, which mostly produced single Ca2+ spikes upon brief stimulation. The spike-like responses were almost invariantly shaped at different agonist doses above a threshold, while response lag markedly decreased with stimulus strength. In AD-MSCs, agonists and IP3 uncaging elicited similar Ca2+ transients but IP3 pulses released Ca2+ without pronounced delay. This suggested that IP3 production was rate-limiting in agonist transduction. In a subpopulation of AD-MSCs, brief agonist pulses elicited Ca2+ bursts crowned by damped oscillations. With properly adjusted parameters of IP3R inhibition by cytosolic Ca2+, the model reproduced such oscillatory Ca2+ responses as well. GEM-GECO1 and R-CEPIA1er, the genetically encoded sensors of cytosolic and reticular Ca2+, respectively, were co-expressed in HEK-293 cells that also responded to agonists in an “all-or-nothing” manner. The experimentally observed Ca2+ signals triggered by ACh in both compartments were properly simulated with the suggested signaling circuit. Thus, the performed modeling of the transduction process provides sufficient theoretical basis for deeper interpretation of experimental findings on agonist-induced Ca2+ signaling in AD-MSCs.

Impact of Maternal Vitamin D Receptor (VDR) Gene Polymorphisms on Spontaneous Preterm Birth (Egyptian Case-Control Study)

Diverse effects of Ba^(2+) and Co^(2+) on fura-2 fluorescence ratio signals (FR, F350/F380) were examined in cultured bovine aortic endothelial cells (BAECs). ATP (3 μM) induced FR oscillations in normal Ca^(2+)-containing solutions. Co^(2+), but not Ba^(2+), inhibited ATP-induced FR oscillations. In Ca^(2+)-free solutions, 3 μM ATP only elicited a single FR transient. When Ba^(2+) replaced Ca^(2+) in the perfusate, 3 μM ATP provoked a distinct FR transient. The maximal rate of rise of the signal was greater than seen without Ba^(2+) and the FR increased and maintained to a high steady level. When external Ca^(2+) was replaced by Co^(2+), ATP-induced FR transient oscillated with a long recovery time constant. In a survey of the single fluorescence at excitation wavelength 350 nm (F350), Co^(2+) decreased the intensity in the presence of ATP and Ca^(2+). Ba^(2+), however, increased F350 and FR as well, especially in Ca^(2+)-free solutions. The results suggest that ATP-induced Ca^(2+) fluctuation is balanced by Ca^(2+) release from internal stores and influx from external Ca^(2+). Ba2+ seems to be permeable through Ca^(2+) channels and competes with Ca^(2+) for the dye. Co^(2+) blocks Ca^(2+) channels but does not inhibit ATP-mediated Ca^(2+) release in cells.

Finely-Tuned Calcium Oscillations in Osteoclast Differentiation and Bone Resorption.

Calcium (Ca2+) plays an important role in regulating the differentiation and function of osteoclasts. Calcium oscillations (Ca oscillations) are well-known phenomena in receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption via calcineurin. Many modifiers are involved in the fine-tuning of Ca oscillations in osteoclasts. In addition to macrophage colony-stimulating factors (M-CSF; CSF-1) and RANKL, costimulatory signaling by immunoreceptor tyrosine-based activation motif-harboring adaptors is important for Ca oscillation generation and osteoclast differentiation. DNAX-activating protein of 12 kD is always necessary for osteoclastogenesis. In contrast, Fc receptor gamma (FcRγ) works as a key controller of osteoclastogenesis especially in inflammatory situation. FcRγ has a cofactor in fine-tuning of Ca oscillations. Some calcium channels and transporters are also necessary for Ca oscillations. Transient receptor potential (TRP) channels are well-known environmental sensors, and TRP vanilloid channels play an important role in osteoclastogenesis. Lysosomes, mitochondria, and endoplasmic reticulum (ER) are typical organelles for intracellular Ca2+ storage. Ryanodine receptor, inositol trisphosphate receptor, and sarco/endoplasmic reticulum Ca2+ ATPase on the ER modulate Ca oscillations. Research on Ca oscillations in osteoclasts has still many problems. Surprisingly, there is no objective definition of Ca oscillations. Causality between Ca oscillations and osteoclast differentiation and/or function remains to be examined.

The Oscillation Amplitude, Not the Frequency of Cytosolic Calcium, Regulates Apoptosis Induction.

SummaryAlthough a rising concentration of cytosolic Ca2+ has long been recognized as an essential signal for apoptosis, the dynamical mechanisms by which Ca2+ regulates apoptosis are not clear yet. To address this, we constructed a computational model that integrates known biochemical reactions and can reproduce the dynamical behaviors of Ca2+-induced apoptosis as observed in experiments. Model analysis shows that oscillating Ca2+ signals first convert into gradual signals and eventually transform into a switch-like apoptotic response. Via the two processes, the apoptotic signaling pathway filters the frequency of Ca2+ oscillations effectively but instead responds acutely to their amplitude. Collectively, our results suggest that Ca2+ regulates apoptosis mainly via oscillation amplitude, rather than frequency, modulation. This study not only provides a comprehensive understanding of how oscillatory Ca2+ dynamically regulates the complex apoptotic signaling network but also presents a typical example of how Ca2+ controls cellular responses through amplitude modulation.