Calcium-induced Release Research Articles

Review on Sodium Calcium Exchanger (Na+-Ca2+X) Electrophysiological Characteristics and its Role in Cardiac Arrhythmias

Cardiac arrhythmias is one of the cardiovascular diseases that cause more death in today’s industrial world. Among the generation of arrhythmias in cardiac myocytes, Early Afterdepolarization (EAD) and Delayed Afterdepolarization (DAD) is most significant one. Sodium -Calcium Exchanger (Na+- Ca2+ X) dysfunction is one of major source of DAD and EAD. Na+- Ca2+ X which exact mechanism is responsible for promoting EAD and DAD still not derived clearly from any cardiac models investigation. It is very important to understand the electrophysiological characteristics of Na+- Ca2+ X and its role in cardiac arrhythmias to clearly detect such arrhythmias effects. In this research article the aim is to build a strong fundamental foundation of Na+- Ca2+ X mechanism in terms of electrophysiological molecular segment (internal structure) arrangements and various Na+- Ca2+ X investigations on cardiac arrhythmias. Na+-Ca2+X is one which belongs to low chemical attraction, high transport capacity and energetically driven by the Na+ ions. The second is an L-type Ca2+ channel, which has a high affinity to Ca2+. Na+-Ca2+X eject nearly thirty (30%) of Ca2+ is required to activate the myofilaments in rabbit, guinea pig and human ventricles and a very small amount (7%) of Ca2+ is required in rat and mouse ventricles. Na+-Ca2+X transport Ca2+ up to 5000 per second (≈6μM/L), which is much greater than SR Ca2+ ATP Ca2+ transport (100 to 150 per second at ≈0.3μM/L). By using voltage patch clamp, Na+-Ca2+X current is measured in guinea pig ventricular myocyte, from that density of Na+-Ca2+X is to be found, 250 to 400 Na+-Ca2+X/μm2 . Na+-Ca2+X current (INaCa) at level [Ca2+]o = 0.50mM, Na+-Ca2+X mechanism of Ca2+ ions removal is very quickly, why because Calcium Induced Calcium Release (CICR) process does increase the cytosolic Ca2+ concentration very early. Enhanced activity of Na+-Ca2+X or increased Na+-Ca2+X channel current reduces the Action Potential (AP) and AP Duration (APD), and triggers rapid firing within cardiac pulmonary veins. Minimum intracellular calcium levels in SR is responsible for systolic dysfunction in failure heart. Similarly it increases the inward Na+-Ca2+X current, life threatening arrhythmias like DAD happen by sudden release of calcium in SR. Finally increased or enhanced or hypertrophy conditions of Na+-Ca2+X is produces EAD and DAD.

The effect of neuronal atrophy on the pathogenesis of Huntington's disease.

Computational models play a pivotal role in the integration of complex biochemical reaction kinetics with the subcellular organization to obtain a deeper understanding of the cellular response to a drug. We have created a realistic 3D reconstruction of a segment of the axon including the endoplasmic reticulum and the subcellular arrangement of molecules involved in calcium homeostasis and calcium-induced calcium release. The dynamics of the system are modeled using MCell4, a stochastic, particle-based Monte Carlo simulator. We studied the effect of cell atrophy on the disease state of Huntington's disease (HD), a fatal neurological disorder that has no cure yet. HD is characterized by motor dysfunction, mental impairment, and cognitive decline. At the molecular level, HD is caused by a mutation in the first exon of the gene encoding the Huntingtin protein resulting in an expanded polyglutamine. Calcium dyshomeostasis is believed to be one of the key causes of HD. In particular, defective RyR function has been reported in HD leading to elevated intracellular Ca2+ levels. One of the drugs currently under development that can potentially be used for HD is a small-molecule drug RycalTM that may act by stabilizing leaky ryanodine receptor (RyR)/Ca2+ release channels that are in development for a severe form of muscle atrophy. Interestingly, our model indicates that even if all receptors and calcium pumps work normally, cell atrophy alone could lead to the “leaky RyR” receptors phenomenon, and will further result in saturation of the calcium buffer, calbindin, and accumulation of intercellular calcium which can be quite toxic to the cell. The computer model that we have built can be further developed to help us understand whether drugs in development or proposed for HD might be worthwhile or not in an effort to treat/cure HD.

Computational design of custom therapeutic cells to correct failing human cardiomyocytes.

Myocardial delivery of non-excitable cells-namely human mesenchymal stem cells (hMSCs) and c-kit+ cardiac interstitial cells (hCICs)-remains a promising approach for treating the failing heart. Recent empirical studies attempt to improve such therapies by genetically engineering cells to express specific ion channels, or by creating hybrid cells with combined channel expression. This study uses a computational modeling approach to test the hypothesis that custom hypothetical cells can be rationally designed to restore a healthy phenotype when coupled to human heart failure (HF) cardiomyocytes. Candidate custom cells were simulated with a combination of ion channels from non-excitable cells and healthy human cardiomyocytes (hCMs). Using a genetic algorithm-based optimization approach, candidate cells were accepted if a root mean square error (RMSE) of less than 50% relative to healthy hCM was achieved for both action potential and calcium transient waveforms for the cell-treated HF cardiomyocyte, normalized to the untreated HF cardiomyocyte. Custom cells expressing only non-excitable ion channels were inadequate to restore a healthy cardiac phenotype when coupled to either fibrotic or non-fibrotic HF cardiomyocytes. In contrast, custom cells also expressing cardiac ion channels led to acceptable restoration of a healthy cardiomyocyte phenotype when coupled to fibrotic, but not non-fibrotic, HF cardiomyocytes. Incorporating the cardiomyocyte inward rectifier K+ channel was critical to accomplishing this phenotypic rescue while also improving single-cell action potential metrics associated with arrhythmias, namely resting membrane potential and action potential duration. The computational approach also provided insight into the rescue mechanisms, whereby heterocellular coupling enhanced cardiomyocyte L-type calcium current and promoted calcium-induced calcium release. Finally, as a therapeutically translatable strategy, we simulated delivery of hMSCs and hCICs genetically engineered to express the cardiomyocyte inward rectifier K+ channel, which decreased action potential and calcium transient RMSEs by at least 24% relative to control hMSCs and hCICs, with more favorable single-cell arrhythmia metrics. Computational modeling facilitates exploration of customizable engineered cell therapies. Optimized cells expressing cardiac ion channels restored healthy action potential and calcium handling phenotypes in fibrotic HF cardiomyocytes and improved single-cell arrhythmia metrics, warranting further experimental validation studies of the proposed custom therapeutic cells.

Disrupted T-tubular network accounts for asynchronous calcium release in MTM1-deficient skeletal muscle.

In mammalian skeletal muscle, the propagation of surface membrane depolarization into the interior of the muscle fibre along the transverse (T) tubular network is essential for the synchronized release of calcium from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) in response to the conformational change in the voltage-sensor dihydropyridine receptors. Deficiency in 3-phosphoinositide phosphatase myotubularin (MTM1) has been reported to disrupt T-tubules, resulting in impaired SR calcium release. Here confocal calcium transients recorded in muscle fibres of MTM1-deficient mice were compared with the results from a model where propagation of the depolarization along the T-tubules was modelled mathematically with disruptions in the network assumed to modify the access and transmembrane resistance as well as the capacitance. If, in simulations, T-tubules were assumed to be partially or completely inaccessible to the depolarization and RyRs at these points to be prime for calcium-induced calcium release, all the features of measured SR calcium release could be reproduced. We conclude that the inappropriate propagation of the depolarization into the fibre interior is the initial critical cause of severely impaired SR calcium release in MTM1 deficiency, while the Ca2+ -triggered opening of RyRs provides an alleviating support to the diseased process. KEY POINTS: Myotubular myopathy is a fatal disease due to genetic deficiency in the phosphoinositide phosphatase MTM1. Although the causes are known and corresponding gene therapy strategies are being developed, there is no mechanistic understanding of the disease-associated muscle function failure. Resolving this issue is of primary interest not only for a fundamental understanding of how MTM1 is critical for healthy muscle function, but also for establishing the related cellular mechanisms most primarily or stringently affected by the disease, which are thus of potential interest as therapy targets. The mathematical modelling approach used in the present work proves that the disease-associated alteration of the plasma membrane invagination network is sufficient to explain the dysfunctions of excitation-contraction coupling, providing the first integrated quantitative framework that explains the associated contraction failure.

Abstract 14073: Hypercalcemia Induced Reversible LBBB

Background: Parathyroid hormone (PTH) and calcitriol are the two hormones responsible for calcium and phosphate homeostasis. PTH exerts its effect on the renal tubular calcium reabsorption and bone resorption. PTH secretion is modulated by calcium acting on the sensitive calcium-sensing receptor on the parathyroid cells. Primary hyperparathyroidism has been linked to hypertension and valvular calcification. Structural changes are also seen including elevated left ventricular mass, diastolic dysfunction and myocardial calcification. Case presentation: A 61-year old female with hyperlipidemia and osteoporosis presented with polyarticular joint pain and diffuse myalgia noted to have elevated PTH of 90 pg/ml and elevated Ca of 10.1 mg/dl. Her calcium levels has ranged from 10.1 to 11.2 mg/dl and her PTH ranged from 90 to 113 pg/ml. She had a negative sestamibi scan, and thyroid ultrasound showed fusiform lesion measuring 0.82 x 0.36 in the right posterior neck suspicious for a parathyroid adenoma with the plan made for resection. The diagnosis at this time was primary hyperparathyroidism induced hypercalcemia. She developed exertional dizziness and was found to have a new LBBB and reduced ejection fraction of 40-45% with a small fixed defect in the septal area which was attributed to the LBBB. She was initiated on metoprolol succinate 25 mg, irbesartan 75 mg, atorvastatin 40 mg and aspirin 81 mg because of the reduced systolic function. SPECT was negative for ischemia with a fixed decreased uptake of the septum. She underwent right lower parathyroid excision on 11/2021 with normalization of calcium to 9.4 mg/dl and PTH to 9 pg/ml with resolution of the LBBB and systolic function in 2/2021 improving to 48%. Discussion: Calcium plays a key role in cardiac excitation, depolarization, repolarization, and also coupling of these signals to mechanical contraction via calcium induced calcium release and myofibril cross linking. In this case, calcium was only mildly elevated to cause a partial conduction defect. Literature is lacking on whether there is a threshold level for calcium that predisposes to varying degrees of conduction disease. This case also highlights the temporal nature of symptom resolution following parathyroid surgery with resolution of the LBBB.

A unique inventory of ion transporters poises the Venus flytrap to fast-propagating action potentials and calcium waves

Since the 19th century, it has been known that the carnivorous Venus flytrap is electrically excitable. Nevertheless, the mechanism and the molecular entities of the flytrap action potential (AP) remain unknown. When entering the electrically excitable stage, the trap expressed a characteristic inventory of ion transporters, among which the increase in glutamate receptor GLR3.6 RNA was most pronounced. Trigger hair stimulation or glutamate application evoked an AP and a cytoplasmic Ca2+ transient that both propagated at the same speed from the site of induction along the entire trap lobe surface. A priming Ca2+ moiety entering the cytoplasm in the context of the AP was further potentiated by an organelle-localized calcium-induced calcium release (CICR)-like system prolonging the Ca2+ signal. While the Ca2+ transient persisted, SKOR K+ channels and AHA H+-ATPases repolarized the AP already. By counting the number of APs and long-lasting Ca2+ transients, the trap directs the different steps in the carnivorous plant's hunting cycle. VIDEO ABSTRACT.

Modality specific roles for metabotropic GABAergic signaling and calcium induced calcium release mechanisms in regulating cold nociception.

Calcium (Ca2+) plays a pivotal role in modulating neuronal-mediated responses to modality-specific sensory stimuli. Recent studies in Drosophila reveal class III (CIII) multidendritic (md) sensory neurons function as multimodal sensors regulating distinct behavioral responses to innocuous mechanical and nociceptive thermal stimuli. Functional analyses revealed CIII-mediated multimodal behavioral output is dependent upon activation levels with stimulus-evoked Ca2+ displaying relatively low vs. high intracellular levels in response to gentle touch vs. noxious cold, respectively. However, the mechanistic bases underlying modality-specific differential Ca2+ responses in CIII neurons remain incompletely understood. We hypothesized that noxious cold-evoked high intracellular Ca2+ responses in CIII neurons may rely upon Ca2+ induced Ca2+ release (CICR) mechanisms involving transient receptor potential (TRP) channels and/or metabotropic G protein coupled receptor (GPCR) activation to promote cold nociceptive behaviors. Mutant and/or CIII-specific knockdown of GPCR and CICR signaling molecules [GABAB-R2, Gαq, phospholipase C, ryanodine receptor (RyR) and Inositol trisphosphate receptor (IP3R)] led to impaired cold-evoked nociceptive behavior. GPCR mediated signaling, through GABAB-R2 and IP3R, is not required in CIII neurons for innocuous touch evoked behaviors. However, CICR via RyR is required for innocuous touch-evoked behaviors. Disruptions in GABAB-R2, IP3R, and RyR in CIII neurons leads to significantly lower levels of cold-evoked Ca2+ responses indicating GPCR and CICR signaling mechanisms function in regulating Ca2+ release. CIII neurons exhibit bipartite cold-evoked firing patterns, where CIII neurons burst during rapid temperature change and tonically fire during steady state cold temperatures. GABAB-R2 knockdown in CIII neurons resulted in disorganized firing patterns during cold exposure. We further demonstrate that application of GABA or the GABAB specific agonist baclofen potentiates cold-evoked CIII neuron activity. Upon ryanodine application, CIII neurons exhibit increased bursting activity and with CIII-specific RyR knockdown, there is an increase in cold-evoked tonic firing and decrease in bursting. Lastly, our previous studies implicated the TRPP channel Pkd2 in cold nociception, and here, we show that Pkd2 and IP3R genetically interact to specifically regulate cold-evoked behavior, but not innocuous mechanosensation. Collectively, these analyses support novel, modality-specific roles for metabotropic GABAergic signaling and CICR mechanisms in regulating intracellular Ca2+ levels and cold-evoked behavioral output from multimodal CIII neurons.

Efficacy of Calcium Chloride Dihydrate Supplementation at ICSI on Patient Outcomes

Background: ICSI typically results in fertilisation rates of >65% however patients may still have low or no fertilisation despite the oocyte and sperm quality appearing normal. After entry of the sperm into the oocyte, a cascade of reactions facilitates calcium release and subsequent oocyte activation. In cases of poor or failed fertilisation it is possible that this cascade fails and therefore different methods of artificial oocyte activation are used to try and induce calcium release. Aim: To determine if calcium chloride dihydrate supplementation in the PVP (5mM) at the time of ICSI (ICSI-Ca) improves fertilisation, utilisation and clinical pregnancy rates compared to ICSI alone, particularly in patients with a history of low fertilisation (<50%). Methods: Retrospective study between 2016-2021 at Monash IVF Victoria on a paired cohort of patients (n=178 patients) where an ICSI cycle was analysed coupled with the subsequent ICSI-Ca cycle. The paired cohort was further subdivided into a low-fertilisation cohort (<50% fertilisation on previous cycles: n=66 patients) compared to the remaining patients with normal-fertilisation ([Formula: see text] 50%; n=122). Exclusion criteria included donor cycles, PGT patients, surgical sperm retrieval, women [Formula: see text] 45 years old, patients with >6 cycles and [Formula: see text] 5 inseminated oocytes. Results: Calcium supplementation increased both fertilisation (28.8% ICSI vs 49.7% ICSI-Ca, p<0.0001) and clinical pregnancy rate (4.9% ICSI vs 25.0% ICSI-Ca: p<0.05) in the low-fertilisation cohort but not in the normal-fertilisation cohort. Interestingly, utilisation rate increased in the normal-fertilisation cohort (32.6% ICSI vs ICSI-Ca: 44.9%, p<0.01) but not in the low-fertilisation cohort, although the number of extra embryos utilised per patient after ICSI-Ca increased in both groups. Conclusion: Calcium supplementation appears not to be a detrimental addition to ICSI and may improve IVF outcomes, particularly for patients with a history of low fertilisation. Further investigations including prospective case matched studies or RCT are required to confirm these findings.

Dynamic S-acylation of the ER-resident protein stromal interaction molecule 1 (STIM1) is required for store-operated Ca2+ entry

Many cell surface stimuli cause calcium release from endoplasmic reticulum (ER) stores to regulate cellular physiology. Upon ER calcium store depletion, the ER-resident protein stromal interaction molecule 1 (STIM1) physically interacts with plasma membrane protein Orai1 to induce calcium release–activated calcium (CRAC) currents that conduct calcium influx from the extracellular milieu. Although the physiological relevance of this process is well established, the mechanism supporting the assembly of these proteins is incompletely understood. Earlier we demonstrated a previously unknown post-translational modification of Orai1 with long-chain fatty acids, known as S-acylation. We found that S-acylation of Orai1 is dynamically regulated in a stimulus-dependent manner and essential for its function as a calcium channel. Here using the acyl resin–assisted capture assay, we show that STIM1 is also rapidly S-acylated at cysteine 437 upon ER calcium store depletion. Using a combination of live cell imaging and electrophysiology approaches with a mutant STIM1 protein, which could not be S-acylated, we determined that the S-acylation of STIM1 is required for the assembly of STIM1 into puncta with Orai1 and full CRAC channel function. Together with the S-acylation of Orai1, our data suggest that stimulus-dependent S-acylation of CRAC channel components Orai1 and STIM1 is a critical mechanism facilitating the CRAC channel assembly and function.

Identification of ASPDH as a novel NAADP-binding protein

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a signaling molecule that can induce calcium release from intracellular acidic stores. However, proteins that bind to NAADP are understudied. Here, we identify aspartate dehydrogenase domain-containing protein (ASPDH) as an NAADP-binding protein through biochemical purification from pig livers. Isothermal titration calorimetry (ITC) experiment using the recombinantly expressed protein shows a 1:1 binding stoichiometry and a Kd of 455 nM between NAADP and mouse ASPDH. In contrast, recombinantly expressed Jupiter microtubule-associated homolog 2 (JPT2) and SM-like protein LSM12, two proteins previously identified as NAADP-receptors, show no binding in ITC experiments.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

Calcium modeling of spine apparatus-containing human dendritic spines demonstrates an "all-or-nothing" communication switch between the spine head and dendrite.

Dendritic spines are highly dynamic neuronal compartments that control the synaptic transmission between neurons. Spines form ultrastructural units, coupling synaptic contact sites to the dendritic shaft and often harbor a spine apparatus organelle, composed of smooth endoplasmic reticulum, which is responsible for calcium sequestration and release into the spine head and neck. The spine apparatus has recently been linked to synaptic plasticity in adult human cortical neurons. While the morphological heterogeneity of spines and their intracellular organization has been extensively demonstrated in animal models, the influence of spine apparatus organelles on critical signaling pathways, such as calcium-mediated dynamics, is less well known in human dendritic spines. In this study we used serial transmission electron microscopy to anatomically reconstruct nine human cortical spines in detail as a basis for modeling and simulation of the calcium dynamics between spine and dendrite. The anatomical study of reconstructed human dendritic spines revealed that the size of the postsynaptic density correlates with spine head volume and that the spine apparatus volume is proportional to the spine volume. Using a newly developed simulation pipeline, we have linked these findings to spine-to-dendrite calcium communication. While the absence of a spine apparatus, or the presence of a purely passive spine apparatus did not enable any of the reconstructed spines to relay a calcium signal to the dendritic shaft, the calcium-induced calcium release from this intracellular organelle allowed for finely tuned "all-or-nothing" spine-to-dendrite calcium coupling; controlled by spine morphology, neck plasticity, and ryanodine receptors. Our results suggest that spine apparatus organelles are strategically positioned in the neck of human dendritic spines and demonstrate their potential relevance to the maintenance and regulation of spine-to-dendrite calcium communication.

Oscillatory calcium release and sustained store-operated oscillatory calcium signaling prevents differentiation of human oligodendrocyte progenitor cells

Endogenous remyelination in demyelinating diseases such as multiple sclerosis is contingent upon the successful differentiation of oligodendrocyte progenitor cells (OPCs). Signaling via the Gαq-coupled muscarinic receptor (M1/3R) inhibits human OPC differentiation and impairs endogenous remyelination in experimental models. We hypothesized that calcium release following Gαq-coupled receptor (GqR) activation directly regulates human OPC (hOPC) cell fate. In this study, we show that specific GqR agonists activating muscarinic and metabotropic glutamate receptors induce characteristic oscillatory calcium release in hOPCs and that these agonists similarly block hOPC maturation in vitro. Both agonists induce calcium release from endoplasmic reticulum (ER) stores and store operated calcium entry (SOCE) likely via STIM/ORAI-based channels. siRNA mediated knockdown (KD) of obligate calcium sensors STIM1 and STIM2 decreased the magnitude of muscarinic agonist induced oscillatory calcium release and attenuated SOCE in hOPCs. In addition, STIM2 expression was necessary to maintain the frequency of calcium oscillations and STIM2 KD reduced spontaneous OPC differentiation. Furthermore, STIM2 siRNA prevented the effects of muscarinic agonist treatment on OPC differentiation suggesting that SOCE is necessary for the anti-differentiative action of muscarinic receptor-dependent signaling. Finally, using a gain-of-function approach with an optogenetic STIM lentivirus, we demonstrate that independent activation of SOCE was sufficient to significantly block hOPC differentiation and this occurred in a frequency dependent manner while increasing hOPC proliferation. These findings suggest that intracellular calcium oscillations directly regulate hOPC fate and that modulation of calcium oscillation frequency may overcome inhibitory Gαq-coupled signaling that impairs myelin repair.

Parental uveitis causes elevated hair loss in offspring of C57BL/6J mice

Our previous study demonstrated that parental uveitis in a susceptible population can cause hair loss and increase the susceptibility to experimental autoimmune uveitis (EAU) in offspring. However, it is unclear whether parental uveitis affects the development of offspring in an EAU-moderate-susceptible population. Herein, moderate-susceptible C57BL/6J mice were immunized with inter-photoreceptor retinoid binding protein (IRBP) 651–670 to develop EAU and were kept together for mating. Gross examination and histopathological changes of the offspring gestated with parental uveitis were observed to evaluate the impact of parental uveitis on the development of the offspring. Differentially expressed genes (DEGs) were screened by RNA sequencing in the affected skin and eyeball of the offspring on postnatal day 27. Adult offspring were injected 75 μg IRBP651-670 to evaluate their susceptibility to EAU. Gross examination in the offspring revealed hair loss on postnatal days 11–31. Histopathological observation showed increased melanin granules and hair follicles of skin in the affected offspring with hair loss. Gene Ontology (GO) analysis in the skin revealed differential expression of genes involved in the mitotic cell cycle, response to endogenous stimulus, hair follicle development, and hair cycle. The DEGs in the skin were predominately associated with the cell cycle and peroxisome proliferator-activated receptor (PPAR) signaling pathway. The GO enrichment analysis in the eyeball showed differential expression of genes involved in the nervous system development, camera-type eye photoreceptor cell differentiation, neuron projection morphogenesis, axon development, and calcium-induced calcium release activity; enriched pathways included the circadian entrainment and glutamatergic synapses. No increased susceptibility to EAU in offspring gestated from parental remitting EAU was observed at a low-dose 75 μg IRBP induction. These results suggested that parental uveitis in a moderate-susceptible population could affect the skin development and DEG profiles of skin and eyeball related to the response to endogenous stimulus, the PPAR signaling pathway, and glutamatergic synapse, which provides the molecular evidence to explain the influence of parental uveitis on offspring development.

Ultrastructural effects of modulating calcium handling in the cardiac intercalated disc

Decreased extracellular calcium in the heart reduces cell-cell adhesion and increases intermembrane spacing in the intercalated disc (ID), particularly within the gap junction adjacent nanodomain known as the perinexus. Disruption of cell-cell adhesion within the perinexus slows cardiac conduction and can lead to arrhythmias. It is unknown whether the perinexus contains calcium-handling proteins or what mechanism(s) may locally regulate calcium levels within these ID nanodomains. L-type calcium channels (Cav1.2) located in T-tubules are the primary mediators of calcium influx and subsequent calcium-induced calcium release from the sarcoplasmic reticulum.

The Thermodynamically Expensive Contribution of Three Calcium Sources to Somatic Release of Serotonin.

The soma, dendrites and axon of neurons may display calcium-dependent release of transmitters and peptides. Such release is named extrasynaptic for occurring in absence of synaptic structures. This review describes the cooperative actions of three calcium sources on somatic exocytosis. Emphasis is given to the somatic release of serotonin by the classical leech Retzius neuron, which has allowed detailed studies on the fine steps from excitation to exocytosis. Trains of action potentials induce transmembrane calcium entry through L-type channels. For action potential frequencies above 5 Hz, summation of calcium transients on individual action potentials activates the second calcium source: ryanodine receptors produce calcium-induced calcium release. The resulting calcium tsunami activates mitochondrial ATP synthesis to fuel transport of vesicles to the plasma membrane. Serotonin that is released maintains a large-scale exocytosis by activating the third calcium source: serotonin autoreceptors coupled to phospholipase C promote IP3 production. Activated IP3 receptors in peripheral endoplasmic reticulum release calcium that promotes vesicle fusion. The Swiss-clock workings of the machinery for somatic exocytosis has a striking disadvantage. The essential calcium-releasing endoplasmic reticulum near the plasma membrane hinders the vesicle transport, drastically reducing the thermodynamic efficiency of the ATP expenses and elevating the energy cost of release.

Magnesium Ions Moderate Calcium-Induced Calcium Release in Cardiac Calcium Release Sites by Binding to Ryanodine Receptor Activation and Inhibition Sites.

Ryanodine receptor channels at calcium release sites of cardiac myocytes operate on the principle of calcium-induced calcium release. In vitro experiments revealed competition of Ca2+ and Mg2+ in the activation of ryanodine receptors (RyRs) as well as inhibition of RyRs by Mg2+. The impact of RyR modulation by Mg2+ on calcium release is not well understood due to the technical limitations of in situ experiments. We turned instead to an in silico model of a calcium release site (CRS), based on a homotetrameric model of RyR gating with kinetic parameters determined from in vitro measurements. We inspected changes in the activity of the CRS model in response to a random opening of one of 20 realistically distributed RyRs, arising from Ca2+/Mg2+ interactions at RyR channels. Calcium release events (CREs) were simulated at a range of Mg2+-binding parameters at near-physiological Mg2+ and ATP concentrations. Facilitation of Mg2+ binding to the RyR activation site inhibited the formation of sparks and slowed down their activation. Impeding Mg-binding to the RyR activation site enhanced spark formation and speeded up their activation. Varying Mg2+ binding to the RyR inhibition site also dramatically affected calcium release events. Facilitation of Mg2+ binding to the RyR inhibition site reduced the amplitude, relative occurrence, and the time-to-end of sparks, and vice versa. The characteristics of CREs correlated dose-dependently with the effective coupling strength between RyRs, defined as a function of RyR vicinity, single-channel calcium current, and Mg-binding parameters of the RyR channels. These findings postulate the role of Mg2+ in calcium release as a negative modulator of the coupling strength among RyRs in a CRS, translating to damping of the positive feedback of the calcium-induced calcium-release mechanism.

Nanoscale Organization, Regulation, and Dynamic Reorganization of Cardiac Calcium Channels.

The architectural specializations and targeted delivery pathways of cardiomyocytes ensure that L-type Ca2+ channels (CaV1.2) are concentrated on the t-tubule sarcolemma within nanometers of their intracellular partners the type 2 ryanodine receptors (RyR2) which cluster on the junctional sarcoplasmic reticulum (jSR). The organization and distribution of these two groups of cardiac calcium channel clusters critically underlies the uniform contraction of the myocardium. Ca2+ signaling between these two sets of adjacent clusters produces Ca2+ sparks that in health, cannot escalate into Ca2+ waves because there is sufficient separation of adjacent clusters so that the release of Ca2+ from one RyR2 cluster or supercluster, cannot activate and sustain the release of Ca2+ from neighboring clusters. Instead, thousands of these Ca2+ release units (CRUs) generate near simultaneous Ca2+ sparks across every cardiomyocyte during the action potential when calcium induced calcium release from RyR2 is stimulated by depolarization induced Ca2+ influx through voltage dependent CaV1.2 channel clusters. These sparks summate to generate a global Ca2+ transient that activates the myofilaments and thus the electrical signal of the action potential is transduced into a functional output, myocardial contraction. To generate more, or less contractile force to match the hemodynamic and metabolic demands of the body, the heart responds to β-adrenergic signaling by altering activity of calcium channels to tune excitation-contraction coupling accordingly. Recent accumulating evidence suggests that this tuning process also involves altered expression, and dynamic reorganization of CaV1.2 and RyR2 channels on their respective membranes to control the amplitude of Ca2+ entry, SR Ca2+ release and myocardial function. In heart failure and aging, altered distribution and reorganization of these key Ca2+ signaling proteins occurs alongside architectural remodeling and is thought to contribute to impaired contractile function. In the present review we discuss these latest developments, their implications, and future questions to be addressed.

Pharmacogenomic Screening of Drug Candidates using Patient-Specific hiPSC-Derived Cardiomyocyte High-Throughput Calcium Imaging.

Calcium imaging is an invaluable technique to detect and characterize calcium flux in cells. The use of calcium dye provides information on the concentration and spatial distribution of calcium. Calcium imaging is a well-established technique to assess the calcium-induced calcium release mechanism in cardiomyocytes. It can also be used to characterize mutations in genes crucial for this mechanism that frequently causes arrhythmia. Here we describe a high-throughput methodology of calcium imaging that records individual calcium transients in more than 10,000 humaninduced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in less than 30min.

Homeostatic calcium fluxes, ER calcium release, SOCE, and calcium oscillations in cultured astrocytes are interlinked by a small calcium toolkit

How homeostatic ER calcium fluxes shape cellular calcium signals is still poorly understood. Here we used dual-color calcium imaging (ER-cytosol) and transcriptome analysis to link candidates of the calcium toolkit of astrocytes with homeostatic calcium signals. We found molecular and pharmacological evidence that P/Q-type channel Cacna1a contributes to depolarization-dependent calcium entry in astrocytes. For stimulated ER calcium release, the cells express the phospholipase Cb3, IP3 receptors Itpr1 and Itpr2, but no ryanodine receptors (Ryr1–3). After IP3-induced calcium release, Stim1/2 – Orai1/2/3 most likely mediate SOCE. The Serca2 (Atp2a2) is the candidate for refilling of the ER calcium store. The cells highly express adenosine receptor Adora1a for IP3-induced calcium release. Accordingly, adenosine induces fast ER calcium release and subsequent ER calcium oscillations. After stimulation, calcium refilling of the ER depends on extracellular calcium. In response to SOCE, astrocytes show calcium-induced calcium release, notably even after ER calcium was depleted by extracellular calcium removal in unstimulated cells. In contrast, spontaneous ER-cytosol calcium oscillations were not fully dependent on extracellular calcium, as ER calcium oscillations could persist over minutes in calcium-free solution. Additionally, cell-autonomous calcium oscillations show a second-long spatial and temporal delay in the signal dynamics of ER and cytosolic calcium. Our data reveal a rather strong contribution of homeostatic calcium fluxes in shaping IP3-induced and calcium-induced calcium release as well as spatiotemporal components of intracellular calcium oscillations.