Assessment of expression of calcium signaling related lncRNAs in epilepsy.

Calcium signaling is a metabolic pathway that is essential in neurons development and can be involved in the pathobiology of epilepsy. We assessed expression of three mRNA coding gene (SLC1A1, SLC25A12, and ATP2B2) and three related long non-coding RNAs (LINC01231:1, lnc-SLC25A12-8:1 and lnc-MTR-1:1) from this pathway in 39 patients with refractory epilepsy and 71 healthy controls. Expression of all genes except for lnc-SLC25A12 was higher in total epileptic cases compared with controls (P values = 0.0002, < 0.0001, < 0.0001, 0.049 and 0.0005 for SLC1A1, SLC25A12, LINC01231, ATP2B2 and lnc-MTR-1, respectively. When we separately compared expression of genes among males and females, SLC1A1, SLC25A12, LINC01231 and lnc-MTR-1 showed up-regulation in male cases compared with male controls. Moreover, expressions of SLC1A1 and SLC25A12 were higher in female cases compared with female controls. Remarkably, SLC25A12 was found to have the highest sensitivity value (= 1) for differentiation of epileptic cases from controls. Moreover, lnc-MTR-1 and lnc-SLC25A12 were sensitive markers for such purpose (sensitivity values = 0.89 and 0.87, respectively). The highest value belonged to LINC01231 with the value of 0.76. Taken together, this study demonstrates dysregulation of calcium-signaling related genes in epileptic patients and suggests these genes as potential biomarkers for epilepsy.

High-throughput assessment identifying major platelet Ca2+ entry pathways via tyrosine kinase-linked and G protein-coupled receptors.

In platelets, elevated cytosolic Ca2+ is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca2+ response consists of Ca2+ mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca2+ entry pathways. Several channels can contribute to the Ca2+ entry, but their relative contribution is unclear upon stimulation of ITAM-linked receptors such as glycoprotein VI (GPVI) and G-protein coupled receptors such as the protease-activated receptors (PAR) for thrombin. We employed a 96-well plate high-throughput assay with Fura-2-loaded human platelets to perform parallel [Ca2+]i measurements in the presence of EGTA or CaCl2. Per agonist condition, this resulted in sets of EGTA, CaCl2 and Ca2+ entry ratio curves, defined by six parameters, reflecting different Ca2+ ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca2+ entry ratio of 3-7. Strikingly, in combination with Ca2+-ATPase inhibition by thapsigargin, the maximal Ca2+ entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca2+ entry. By pharmacological blockage of specific Ca2+ channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca2+ entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na+/Ca2+ exchange (NCE) > P2×1 (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca2+ carriers regulating GPVI- and PAR-induced Ca2+ entry in human platelets.

Assessment of a 3D neural spheroid model to detect pharmaceutical-induced neurotoxicity.

Drug-induced neurotoxicity is a leading cause of safety-related attrition for therapeutics in clinical trials, often driven by poor predictivity of preclinical in vitro and in vivo models of neurotoxicity. Over a dozen different iPSC-derived 3D spheroids have been described in recent years, but their ability to predict neurotoxicity in patients has not been evaluated nor compared with the predictive power of nonclinical species. To assess the predictive capabilities of human iPSC-derived neural spheroids (microBrains), we used 84 structurally diverse pharmaceuticals with robust clinical and pre-clinical datasets with varying degrees of seizurogenic and neurodegenerative liability. Drug-induced changes in neural viability and phenotypic calcium bursts were assessed using 7 endpoints based on calcium oscillation profiles and cel-lular ATP levels. These endpoints, normalized by therapeutic exposure, were used to build logistic regression models to establish endpoint cutoffs and evaluate probability for clinical neurotoxicity. The neurotoxicity score calculated from the logistic regression model could distinguish neurotoxic from non-neurotoxic clinical molecules with a specificity as high as 93.33% and a sensitivity of 53.49%, demonstrating a very low false positive rate for the prediction of seizures, convulsions, and neurodegeneration. In contrast, nonclinical species showed a higher sensitivity (75%) but much lower specificity (30.4%). The neural spheroids demonstrated higher likelihood ratio positive and inverse likelihood ratio neg-ative values compared with nonclinical safety studies. This assay has the potential to be used as a predictive assay to detect neurotoxicity in early drug discovery, aiding in the early identification of compounds that eventually may fail due to neurotoxicity.

Synthesis and Biological Assessment of 4,1-Benzothiazepines with Neuroprotective Activity on the Ca2+ Overload for the Treatment of Neurodegenerative Diseases and Stroke.

In excitable cells, mitochondria play a key role in the regulation of the cytosolic Ca2+ levels. A dysregulation of the mitochondrial Ca2+ buffering machinery derives in serious pathologies, where neurodegenerative diseases highlight. Since the mitochondrial Na+/Ca2+ exchanger (NCLX) is the principal efflux pathway of Ca2+ to the cytosol, drugs capable of blocking NCLX have been proposed to act as neuroprotectants in neuronal damage scenarios exacerbated by Ca2+ overload. In our search of optimized NCLX blockers with augmented drug-likeness, we herein describe the synthesis and pharmacological characterization of new benzothiazepines analogues to the first-in-class NCLX blocker CGP37157 and its further derivative ITH12575, synthesized by our research group. As a result, we found two new compounds with an increased neuroprotective activity, neuronal Ca2+ regulatory activity and improved drug-likeness and pharmacokinetic properties, such as clog p or brain permeability, measured by PAMPA experiments.

High-Dose Epinephrine Enhances Platelet Aggregation at the Expense of Procoagulant Activity.

Platelet activation is characterized by shape change, granule secretion, activation of fibrinogen receptor (glycoprotein IIb/IIIa) sustaining platelet aggregation, and externalization of negatively charged aminophospholipids contributing to platelet procoagulant activity. Epinephrine (EPI) alone is a weak platelet activator. However, it is able to potentiate platelet activation initiated by other agonists. In this work, we investigated the role of EPI in the generation of procoagulant platelets. Human platelets were activated with convulxin (CVX), thrombin (THR) or protease-activated receptor (PAR) agonists, EPI, and combination thereof. Platelet aggregation was assessed by light transmission aggregometry or with PAC-1 binding by flow cytometry. Procoagulant collagen-and-THR (COAT) platelets, induced by combined activation with CVX-and-THR, were visualized by flow cytometry as Annexin-V-positive and PAC-1-negative platelets. Cytosolic calcium fluxes were monitored by flow cytometry using Fluo-3 indicator. EPI increased platelet aggregation induced by all agonist combinations tested. On the other hand, EPI dose-dependently reduced the formation of procoagulant COAT platelets generated by combined CVX-and-THR activation. We observed a decreased Annexin-V-positivity and increased binding of PAC-1 with the triple activation (CVX + THR + EPI) compared with CVX + THR. Calcium mobilization with triple activation was decreased with the higher EPI dose (1,000 µM) compared with CVX + THR calcium kinetics. In conclusion, when platelets are activated with CVX-and-THR, the addition of increasing concentrations of EPI (triple stimulation) modulates platelet response reducing cytosolic calcium mobilization, decreasing procoagulant activity, and enhancing platelet aggregation.

Presynaptic endoplasmic reticulum regulates short-term plasticity in hippocampal synapses.

Short-term plasticity preserves a brief history of synaptic activity that is communicated to the postsynaptic neuron. This is primarily regulated by a calcium signal initiated by voltage dependent calcium channels in the presynaptic terminal. Imaging studies of CA3-CA1 synapses reveal the presence of another source of calcium, the endoplasmic reticulum (ER) in all presynaptic terminals. However, the precise role of the ER in modifying STP remains unexplored. We performed in-silico experiments in synaptic geometries based on reconstructions of the rat CA3-CA1 synapses to investigate the contribution of ER. Our model predicts that presynaptic ER is critical in generating the observed short-term plasticity profile of CA3-CA1 synapses and allows synapses with low release probability to operate more reliably. Blocking the ER lowers facilitation in a manner similar to what has been previously characterized in animal models of Alzheimer's disease and underscores the important role played by presynaptic stores in normal function.

Optical Assessment of Nociceptive TRP Channel Function at the Peripheral Nerve Terminal.

Free nerve endings are key structures in sensory transduction of noxious stimuli. In spite of this, little is known about their functional organization. Transient receptor potential (TRP) channels have emerged as key molecular identities in the sensory transduction of pain-producing stimuli, yet the vast majority of our knowledge about sensory TRP channel function is limited to data obtained from in vitro models which do not necessarily reflect physiological conditions. In recent years, the development of novel optical methods such as genetically encoded calcium indicators and photo-modulation of ion channel activity by pharmacological tools has provided an invaluable opportunity to directly assess nociceptive TRP channel function at the nerve terminal.

Filamin A Is Required for NK Cell Cytotoxicity at the Expense of Cytokine Production via Synaptic Filamentous Actin Modulation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions.

Interactions between calmodulin and neurogranin govern the dynamics of CaMKII as a leaky integrator.

Calmodulin-dependent kinase II (CaMKII) has long been known to play an important role in learning and memory as well as long term potentiation (LTP). More recently it has been suggested that it might be involved in the time averaging of synaptic signals, which can then lead to the high precision of information stored at a single synapse. However, the role of the scaffolding molecule, neurogranin (Ng), in governing the dynamics of CaMKII is not yet fully understood. In this work, we adopt a rule-based modeling approach through the Monte Carlo method to study the effect of Ca2+ signals on the dynamics of CaMKII phosphorylation in the postsynaptic density (PSD). Calcium surges are observed in synaptic spines during an EPSP and back-propagating action potential due to the opening of NMDA receptors and voltage dependent calcium channels. Using agent-based models, we computationally investigate the dynamics of phosphorylation of CaMKII monomers and dodecameric holoenzymes. The scaffolding molecule, Ng, when present in significant concentration, limits the availability of free calmodulin (CaM), the protein which activates CaMKII in the presence of calcium. We show that Ng plays an important modulatory role in CaMKII phosphorylation following a surge of high calcium concentration. We find a non-intuitive dependence of this effect on CaM concentration that results from the different affinities of CaM for CaMKII depending on the number of calcium ions bound to the former. It has been shown previously that in the absence of phosphatase, CaMKII monomers integrate over Ca2+ signals of certain frequencies through autophosphorylation (Pepke et al, Plos Comp. Bio., 2010). We also study the effect of multiple calcium spikes on CaMKII holoenzyme autophosphorylation, and show that in the presence of phosphatase, CaMKII behaves as a leaky integrator of calcium signals, a result that has been recently observed in vivo. Our models predict that the parameters of this leaky integrator are finely tuned through the interactions of Ng, CaM, CaMKII, and PP1, providing a mechanism to precisely control the sensitivity of synapses to calcium signals. Author Summary not valid for PLOS ONE submissions.

Low-cost calcium fluorometry for long-term nanoparticle studies in living cells.

Calcium fluorometry is critical to determine cell homeostasis or to reveal communication patterns in neuronal networks. Recently, characterizing calcium signalling in neurons related to interactions with nanomaterials has become of interest due to its therapeutic potential. However, imaging of neuronal cell activity under stable physiological conditions can be either very expensive or limited in its long-term capability. Here, we present a low-cost, portable imaging system for long-term, fast-scale calcium fluorometry in neurons. Using the imaging system, we revealed temperature-dependent changes in long-term calcium signalling in kidney cells and primary cortical neurons. Furthermore, we introduce fast-scale monitoring of synchronous calcium activity in neuronal cultures in response to nanomaterials. Through graph network analysis, we found that calcium dynamics in neurons are temperature-dependent when exposed to chitosan-coated nanoparticles. These results give new insights into nanomaterial-interaction in living cultures and tissues based on calcium fluorometry and graph network analysis.

Distinct mechanisms mediate pacemaker dysfunction associated with catecholaminergic polymorphic ventricular tachycardia mutations: Insights from computational modeling.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced ventricular arrhythmia associated with rhythm disturbance and impaired sinoatrial node cell (SANC) automaticity (pauses). Mutations associated with dysfunction of Ca2+-related mechanisms have been shown to be present in CPVT. These dysfunctions include impaired Ca2+ release from the ryanodine receptor (i.e., RyR2R4496C mutation) or binding to calsequestrin 2 (CASQ2). In SANC, Ca2+ signaling directly and indirectly mediates pacemaker function. We address here the following research questions: (i) what coupled-clock mechanisms and pathways mediate pacemaker mutations associated with CPVT in basal and in response to ß-adrenergic stimulation? (ii) Can different mechanisms lead to the same CPVT-related pacemaker pauses? (iii) Can the mutation-induced deteriorations in SANC function be reversed by drug intervention or gene manipulation? We used a numerical model of mice SANC that includes membrane and intracellular mechanisms and their interconnected signaling pathways. In the basal state of RyR2R4496C SANC, the model predicted that the Na+-Ca2+ exchanger current (INCX) and T-type Ca2+ current (ICaT) mediate between changes in Ca2+ signaling and SANC dysfunction. Under ß-adrenergic stimulation, changes in cAMP-PKA signaling and the sodium currents (INa), in addition to INCX and ICaT, mediate between changes in Ca2+ signaling and SANC automaticity pauses. Under basal conditions in Casq2-/-, the same mechanisms drove changes in Ca2+ signaling and subsequent pacemaker dysfunction. However, SANC automaticity pauses in response to ß-AR stimulation were mediated by ICaT and INa. Taken together, distinct mechanisms can lead to CPVT-associated SANC automaticity pauses. In addition, we predict that specifically increasing SANC cAMP-PKA activity by either a pharmacological agent (IBMX, a phosphodiesterase (PDE) inhibitor), gene manipulation (overexpression of adenylyl cyclase 1/8) or direct manipulation of the SERCA phosphorylation target through changes in gene expression, compensate for the impairment in SANC automaticity. These findings suggest new insights for understanding CPVT and its therapeutic approach.

Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling.

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

Assessment of doxorubicin-induced remodeling of Ca2+ signaling and associated Ca2+ regulating proteins in MDA-MB-231 breast cancer cells.

Triple-negative breast cancers (TNBC) are often associated with high relapse rates, despite treatment with chemotherapy agents such as doxorubicin. A better understanding of the signaling and molecular changes associated with doxorubicin may provide novel insights into strategies to enhance treatment efficacy. Calcium signaling is involved in many pathways influencing the efficacy of chemotherapy agents such as proliferation and cell death. However, there are a limited number of studies exploring the effect of doxorubicin on calcium signaling in TNBC. In this study, MDA-MB-231 triple-negative, basal breast cancer cells stably expressing the genetically-encoded calcium indicator GCaMP6m (GCaMP6m-MDA-MB-231) were used to define alterations in calcium signaling. The effects of doxorubicin in GCaMP6m-MDA-MB-231 cells were determined using live cell imaging and fluorescence microscopy. Changes in mRNA levels of specific calcium regulating proteins as a result of doxorubicin treatment were also assessed using real time qPCR. Doxorubicin (1 µM) produced alterations in intracellular calcium signaling, including enhancing the sensitivity of MDA-MB-231 cells to ATP stimulation and prolonging the recovery time after store-operated calcium entry. Upregulation in mRNA levels of ORAI1, TRPC1, SERCA1, IP3R2 and PMCA2 with doxorubicin 1 µM treatment was also observed. Doxorubicin treatment is associated with specific remodeling in calcium signaling in MDA-MB-231 cells, with associated changes in mRNA levels of specific calcium-regulating proteins.

Simultaneous assessment of calcium handling and contractility dynamics in isolated ventricular myocytes of a rat model of post-acute isoproterenol-induced cardiomyopathy.

Stress-induced cardiomyopathy (SIC) results from a profound catecholaminergic surge during strong emotional or physical stress. SIC is characterized by acute left ventricular apex hypokinesia, in the absence of coronary arteries occlusion, and can lead to arrhythmias and acute heart failure. Although, most SIC patients recover, the process could be slow, and recurrence or death may occur. Despite that the SIC common denominator is a large catecholamine discharge, the pathophysiological mechanism is incompletely understood. It is thought that catecholamines have direct cytotoxicity on apical ventricular myocytes (VM), which have the highest ß-adrenergic receptors density, and whose overstimulation might cause acute Ca2+ overload and oxidative stress, causing death in some VM and stunning others. Rodents receiving acute isoproterenol (ISO) overdose (OV) mimic SIC development, however, they have not been used to simultaneously assess Ca2+ handling and contractility status in isolated VM, which might explain ventricular hypokinesia. Therefore, treating rats with a single ISO-OV (67 mg/kg body weight), we sought out to characterize, with confocal imaging, Ca2+ and shortening dynamics in Fluo-4-loaded VM, during the early (1-5 days) and late post-acute phases (15 days). We found that ISO-OV VM showed contractile dysfunction; blunted shortening with slower force development and relaxation. These correlated with Ca2+ mishandling; blunted Ca2+ transient, with slower time to peak and SR Ca2+ recovery. SR Ca2+ content was low, nevertheless, diastolic Ca2+ sparks were more frequent, and their duration increased. Contractility and Ca2+ dysfunction aggravated or remained altered over time, explaining slow recovery. We conclude that diminished VM contractility is the main determinant of ISO-OV hypokinesia and is mostly related to Ca2+ mishandling.

Rethinking calcium profiles around single channels: the exponential and periodic calcium nanodomains.

Many fundamental calcium-dependent physiological processes are triggered by high local calcium levels that are established around the sites of calcium entry into the cell (channels). They are dubbed as calcium nanodomains but their exact profiles are still elusive. The concept of calcium nanodomains stems from a linear model of calcium diffusion and is only valid when calcium increases are smaller than the concentration of cytoplasmic buffers. Recent data indicates that much higher calcium levels cause buffer saturation. Therefore, I sought explicit solutions of a nonlinear reaction-diffusion model and found a dichotomous solution. For small fluxes, the steady state calcium profile is quasi-exponential, and when calcium exceeds buffer concentration a spatial periodicity appears. Analytical results are supported by Monte-Carlo simulations. I also imaged 1D- and radial calcium distributions around single α-synuclein channels in cell-free conditions. Measured Ca profiles are consistent with theoretical predictions. I propose that the periodic calcium patterns may well arise under certain conditions and their specific functional role has to be established.

Absinthin, an agonist of the bitter taste receptor hTAS2R46, uncovers an ER-to-mitochondria Ca2+-shuttling event.

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extraoral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 µm) in ASM cells does not induce Ca2+ signals but reduces histamine-induced cytosolic Ca2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca2+ probes targeted to the cytosol, subplasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca2+ rises and simultaneously increases Ca2+ influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3ß-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the ß2 agonist salbutamol also could induce Ca2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca2+ uptake, adding a further facet to the ability of cells to encode complex Ca2+ signals.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Assessment of the calcium releasing machinery in oocytes that failed to fertilize after conventional ICSI and assisted oocyte activation.

RESEARCH QUESTION: Can oocyte-related activation deficiencies be evaluated in oocytes that failed to fertilize after intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA)? DESIGN: Evaluation of the spindle-chromosome complexes and intracellular distribution of inositol trisphosphate type 1 receptors (IP3R1) in in-vitro matured (IVM) and failed-to-fertilize oocytes from patients undergoing AOA. Assessment of the oocyte-related Ca2+ releasing capacity in response to Ca2+ ionophores and sperm microinjection in oocytes that failed to fertilize after ICSI or ICSI-AOA. RESULTS: IVM oocytes from patients undergoing conventional ICSI (control) and ICSI-AOA (study group) revealed a similar normalcy of spindle-chromosome complexes and distribution patterns of IP3R1. Failed-to-fertilize oocytes from both groups showed significant differences in proportion of normal or abnormal spindle-chromosome complex conformations. However, migration of IP3R1 was identified in a higher proportion of failed-to-fertilize oocytes after ICSI-AOA than after conventional ICSI. It was further observed that oocytes which failed to fertilize, either after ICSI or ICSI-AOA, mostly retain their capacity to respond to stimuli such as exposure to Ca2+ ionophores or to sperm microinjection. CONCLUSIONS: Evaluation of spindle-chromosome normalcy and distribution of IP3R1 does not help identify the presence of Ca2+ releasing deficiencies in these oocytes. However, oocyte Ca2+ analysis adds value in identifying Ca2+ releasing incapacity of oocytes that failed to fertilize after ICSI or ICSI-AOA. Some patients experiencing fertilization failure after ICSI-AOA present with a suspected activation deficiency downstream of the Ca2+ machinery, which cannot be overcome by ICSI-AOA based on the use of Ca2+ ionophores.

Mechanisms of sex differences in atrial fibrillation: role of hormones and differences in electrophysiology, structure, function, and remodelling.

Atrial fibrillation (AF) is the clinically most prevalent rhythm disorder with large impact on quality of life and increased risk for hospitalizations and mortality in both men and women. In recent years, knowledge regarding epidemiology, risk factors, and patho-physiological mechanisms of AF has greatly increased. Sex differences have been identified in the prevalence, clinical presentation, associated comorbidities, and therapy outcomes of AF. Although it is known that age-related prevalence of AF is lower in women than in men, women have worse and often atypical symptoms and worse quality of life as well as a higher risk for adverse events such as stroke and death associated with AF. In this review, we evaluate what is known about sex differences in AF mechanisms-covering structural, electrophysiological, and hormonal factors-and underscore areas of knowledge gaps for future studies. Increasing our understanding of mechanisms accounting for these sex differences in AF is important both for prognostic purposes and the optimization of (targeted, mechanism-based, and sex-specific) therapeutic approaches.

Endocannabinoid and nitric oxide systems of the hypothalamic paraventricular nucleus mediate effects of NPY on energy expenditure.

OBJECTIVE: Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN. METHODS: Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis. RESULTS: We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure. CONCLUSION: Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.