Cytoplasmic Ca2 Research Articles

Loss of physical contact in space alters the dopamine system in C. elegans

SummaryProgressive neuromuscular decline in microgravity is a prominent health concern preventing interplanetary human habitation. We establish functional dopamine-mediated impairments as a consistent feature across multiple spaceflight exposures and during simulated microgravity in C. elegans. Animals grown continuously in these conditions display reduced movement and body length. Loss of mechanical contact stimuli in microgravity elicits decreased endogenous dopamine and comt-4 (catechol-O-methyl transferase) expression levels. The application of exogenous dopamine reverses the movement and body length defects caused by simulated microgravity. In addition, increased physical contact made comt-4 and dopamine levels rise. It also increased muscular cytoplasmic Ca2+ firing. In dop-3 (D2-like receptor) mutants, neither decrease in movement nor in body length were observed during simulated microgravity growth. These results strongly suggest that targeting the dopamine system through manipulation of the external environment (contact stimuli) prevents muscular changes and is a realistic and viable treatment strategy to promote safe human deep-space travel.

Calcium-Mediated Calpain Activation and Microtubule Dissociation in Cell Model of Hereditary Sensory Neuropathy Type-1 Expressing V144D SPTLC1 Mutation.

Hereditary sensory neuropathy type 1A (HSN1A) is an autosomal, dominantly inherited peripheral neuropathy caused by mutations in serine palmitoyl transferase long chain 1 (SPTLC1), involved in the de novo synthesis of sphingolipids. We have previously reported calcium imbalance, as well as mitochondrial and ER stress in both HSN1 patient lymphoblasts and a transiently transfected cell model. In this study, we investigated the role of the Ca2+-activated protease calpain in destabilizing the cell cytoskeleton, by examining calpain activity in SH-SY5Y cells overexpressing the V144D mutant and changes in microtubule-associated proteins (MAP). Intramitochondrial Ca2+ was found to be significantly depleted and cytoplasmic Ca2+ increased in the V144D mutant. Subsequently, calpain and proteasome activity were increased and calpain substrates, microtubule associated proteins MAP2, and tau were significantly reduced in the microtubule fraction of the mutant. Significant changes were also found in motor proteins dynein and KIF2A detected in the microtubule fraction of cells overexpressing the V144D mutation. There was also a reduction in anterograde and retrograde mitochondrial transport velocities in the V144D mutant. These findings strongly implicate cytoskeletal aberration caused by Ca2+ dysregulation and subsequent loss of microtubule transport functions as the cause of axonal dying back that is characteristic of HSN1.

ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling.

ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated ORAI1 and STIM1 knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, ORAI1 deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in ORAI1 knockout cells, likely because of reduced expression of the Ca2+-dependent transcription factors of the AP-1 family and MEF2C Accordingly, modulation of homeostatic Ca2+ concentration by pretreatment with ORAI1 blocker or agonist could influence baseline IFNB expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.

Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation.

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca2+-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca2+]i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca2+-ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca2+-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.

Deep learning models for image and data processes of intracellular calcium ions

Intracellular calcium ion (Ca2+) in cytoplasm as an intracellular second messenger is involved in almost all important cellular activities of organisms. Generally its concentration ([Ca2+]i) is tested by live imaging followed image and data processes, in which much tedious and subjective manual work is involved. Here we show a computational approach of Deep Calcium following the principles of deep learning to predict the cytoplasmic Ca2+ ranges and calcium peaks in calcium curve of objective cells. To validate Deep Calcium, chondrocytes, bone marrow stromal cells (BMSCs) and osteoblastic like cells (MC3T3-E1) from both the tissue and cell samples as well as from spontaneous and mechanical stimulated calcium response patterns are used. The good performance comparing with other relative machine learning models, as well as consistency biological results with human experts are demonstrated. Deep Calcium provides references for other image and data processes of intracellular range determination and curve peak identification.

A Stochastic Spatiotemporal Model of Rat Ventricular Myocyte Calcium Dynamics Demonstrated Necessary Features for Calcium Wave Propagation.

Calcium (Ca2+) plays a central role in the excitation and contraction of cardiac myocytes. Experiments have indicated that calcium release is stochastic and regulated locally suggesting the possibility of spatially heterogeneous calcium levels in the cells. This spatial heterogeneity might be important in mediating different signaling pathways. During more than 50 years of computational cell biology, the computational models have been advanced to incorporate more ionic currents, going from deterministic models to stochastic models. While periodic increases in cytoplasmic Ca2+ concentration drive cardiac contraction, aberrant Ca2+ release can underly cardiac arrhythmia. However, the study of the spatial role of calcium ions has been limited due to the computational expense of using a three-dimensional stochastic computational model. In this paper, we introduce a three-dimensional stochastic computational model for rat ventricular myocytes at the whole-cell level that incorporate detailed calcium dynamics, with (1) non-uniform release site placement, (2) non-uniform membrane ionic currents and membrane buffers, (3) stochastic calcium-leak dynamics and (4) non-junctional or rogue ryanodine receptors. The model simulates spark-induced spark activation and spark-induced Ca2+ wave initiation and propagation that occur under conditions of calcium overload at the closed-cell condition, but not when Ca2+ levels are normal. This is considered important since the presence of Ca2+ waves contribute to the activation of arrhythmogenic currents.

Low-frequency oscillations of murine skin microcirculations and periodic changes of [Ca2+ ]i and [NO]i levels in murine endotheliocytes: An effect of provocative tests.

The five frequency intervals of skin blood oscillation were described: cardiac, respiratory, myogenic, neurogenic, and endothelial. The endothelial interval is derived into NO-independent and NO-dependent. The exact molecular, cell, or systemic mechanisms of endothelial oscillations generation are unclear. We proposed that oscillations of Ca2+ and NO in endotheliocytes may be possible sources of skin blood perfusion (SBP) oscillations in endothelial interval. To examine our hypothesis we compared the oscillations of cytoplasmic Ca2+ and NO ([Ca2+ ]i and [NO]i ) concentration in cultured murine microvascular endotheliocytes and SBP oscillations in mice. Local heating test and model hypoxia were used as tools to evaluate an interconnection of studied parameters. [Ca2+ ]i and [NO]i were measured simultaneously by Fura-2 AM and DAF-FM. The SBP was measured by laser Doppler flowmetry. The [Ca2+ ]i and [NO]i oscillations at 0.005-0.01 Hz were observed in endotheliocytes, that coincides the ranges of NO-independent endothelial interval. Heating decreased amplitude of [Ca2+ ]i and [NO]i oscillations in cells in NO-independent endothelial interval, while amplitudes of SBP oscillations increased in NO-independent and NO-dependent intervals. Hypoxia reduced the [NO]i oscillations amplitude. Heating test during hypoxia increased NO-independent endothelial SBP oscillations and decreased myogenic ones, did not effect on [NO]i oscillations, and shifted [Ca2+ ]i oscillations peak from 0.005-0.01 Hz to 0.01-0.018 Hz. We observed the [Ca2+ ]i and [NO]i oscillations synchronization within a cell and between cells for the first time. Heating abolished these synchronizations. Therefore low-frequency [Ca2+ ]i and [NO]i oscillations in endotheliocytes may be considered as modulators of low-frequency endothelial SBP oscillations.

Piezo1 controls cell volume and migration by modulating swelling-activated chloride current through Ca2+ influx.

Regulatory volume decrease (RVD), a homeostatic process responsible for the re-establishment of the original cell volume upon swelling, is critical in controlling several functions, including migration. RVD is mainly sustained by the swelling-activated Cl- current (ICl,swell ), which can be modulated by cytoplasmic Ca2+ . Cell swelling also activates mechanosensitive channels, including the ubiquitously expressed Ca2+ -permeable channel Piezo1. We hypothesized that, by controlling cytoplasmic Ca2+ and in turn ICl,swell , Piezo1 is involved in the fine regulation of RVD and cell migration. We compared RVD and ICl,swell in wild-type (WT) HEK293T cells, which express endogenous levels of Piezo1, and in cells overexpressing (OVER) or knockout (KO) for Piezo1. Compared to WT, RVD was markedly increased in OVER, while virtually absent in KO cells. Consistently, ICl,swell amplitude was highest in OVER and lowest in KO cells, with WT cells displaying an intermediate level, suggesting a Ca2+ -dependent modulation of the current by Piezo1 channels. Indeed, in the absence of external Ca2+ , ICl,swell in both WT and OVER cells, as well as the RVD probed in OVER cells, were significantly lower than in the presence of Ca2+ and no longer different compared to KO cells. However, the Piezo-mediated Ca2+ influx was ineffective in enhancing ICl,swell in the absence of releasable Ca2+ from intracellular stores. The different expression levels of Piezo1 affected also cell migration which was strongly enhanced in OVER, while reduced in KO cells, as compared to WT. Taken together, our data indicate that Piezo1 controls RVD and migration in HEK293T cells by modulating ICl,swell through Ca2+ influx.

Mipsagargin: The Beginning-Not the End-of Thapsigargin Prodrug-Based Cancer Therapeutics.

Søren Brøgger Christensen isolated and characterized the cell-penetrant sesquiterpene lactone Thapsigargin (TG) from the fruit Thapsia garganica. In the late 1980s/early 1990s, TG was supplied to multiple independent and collaborative groups. Using this TG, studies documented with a large variety of mammalian cell types that TG rapidly (i.e., within seconds to a minute) penetrates cells, resulting in an essentially irreversible binding and inhibiting (IC50~10 nM) of SERCA 2b calcium uptake pumps. If exposure to 50–100 nM TG is sustained for >24–48 h, prostate cancer cells undergo apoptotic death. TG-induced death requires changes in the cytoplasmic Ca2+, initiating a calmodulin/calcineurin/calpain-dependent signaling cascade that involves BAD-dependent opening of the mitochondrial permeability transition pore (MPTP); this releases cytochrome C into the cytoplasm, activating caspases and nucleases. Chemically unmodified TG has no therapeutic index and is poorly water soluble. A TG analog, in which the 8-acyl groups is replaced with the 12-aminododecanoyl group, afforded 12-ADT, retaining an EC50 for killing of <100 nM. Conjugation of 12-ADT to a series of 5–8 amino acid peptides was engineered so that they are efficiently hydrolyzed by only one of a series of proteases [e.g., KLK3 (also known as Prostate Specific Antigen); KLK2 (also known as hK2); Fibroblast Activation Protein Protease (FAP); or Folh1 (also known as Prostate Specific Membrane Antigen)]. The obtained conjugates have increased water solubility for systemic delivery in the blood and prevent cell penetrance and, thus, killing until the TG-prodrug is hydrolyzed by the targeting protease in the vicinity of the cancer cells. We summarize the preclinical validation of each of these TG-prodrugs with special attention to the PSMA TG-prodrug, Mipsagargin, which is in phase II clinical testing.

L-type calcium channel subtypes mediating hypothalamic dysfunction caused by amyloid-beta.

In Alzheimer's disease (AD), non-cognitive manifestations attributable to hypothalamic dysfunction such as systemic metabolic deficits can occur prior to the cognitive decline (Ishii, M. and Iadecola C., Cell Metabolism 22:761,2015). We previously found in Tg2576 mice overexpressing the Swedish mutation of amyloid precursor protein that amyloid-beta pathology can cause dysfunction in hypothalamic arcuate neuropeptide Y (NPY) neurons, associated with low adiposity and low plasma leptin levels prior to memory decline or plaque formation (Ishii, M. et al., J Neuroscience 34:9096,2014). Furthermore, the hypothalamic arcuate NPY neuronal dysfunction was caused by amyloid-beta modulating low-threshold activated L-type calcium channels (Ishii, M. et al., J Neuroscience 39:8816,2019). However, which of the two major brain L-type calcium channel subtypes Cav 1.2 or Cav 1.3 is involved has not been established. To examine the role of Cav 1.2, we used the Cre-lox system in genetic mouse models to knock-out Cav 1.2 in hypothalamic arcuate NPY neurons (Cav 1.2 cKO). Cav 1.2 cKO mice were bred to NPY-GFP and Tg2576 mice. Two to 3-month-old male or female mice were used for all studies. Voltage-gated L-type calcium currents from hypothalamic slices containing arcuate NPY neurons were recorded using whole-cell voltage-clamp. Cytoplasmic Ca2+ levels were measured ratiometrically by loading Fura-2AM in dissociated arcuate NPY neurons and recording the emitted fluorescence using an inverted fluorescent microscope. Exogenous oligomeric amyloid-β42 caused a significant increase in cytoplasmic Ca2+ levels and L-type calcium currents in arcuate NPY neurons to a similar extent in both Cav 1.2 cKO and wild-type (WT) littermate mice. These effects of amyloid-β42 were reversed by the L-type calcium channel blocker nimodipine. In vivo, Cav 1.2 cKO mice bred to Tg2576 mice had similar body weight deficits compared to Tg2576 mice without deletion of Cav 1.2 in the arcuate NPY neurons. These results suggest that Cav 1.2 does not have a significant role in amyloid-beta mediated dysfunction of hypothalamic arcuate NPY neurons and that Cav 1.3 is likely the major target of amyloid-beta in these neurons. Future studies modulating Cav 1.3 in hypothalamic neurons will help elucidate the mechanisms underlying hypothalamic dysfunction caused by amyloid-beta pathology and its therapeutic potential in AD.

Use of Ore-Derived Humic Acids With Diverse Chemistries to Elucidate Structure-Activity Relationships (SAR) of Humic Acids in Plant Phenotypic Expression.

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Excessive Accumulation of Intracellular Ca2+ After Acute Exercise Potentiated Impairment of T-cell Function.

Ca2+ is an important intracellular second messenger known to regulate several cellular functions. This research aimed to investigate the mechanisms of exercise-induced immunosuppression by measuring intracellular calcium levels, Ca2+-regulating gene expression, and agonist-evoked proliferation of murine splenic T lymphocytes. Mice were randomly assigned to the control, sedentary group (C), and three experimental groups, which performed a single bout of intensive and exhaustive treadmill exercise. Murine splenic lymphocytes were separated by density-gradient centrifugation immediately (E0), 3h (E3), and 24h after exercise (E24). Fura-2/AM was used to monitor cytoplasmic free Ca2+ concentration in living cells. The combined method of carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry was used for the detection of T cell proliferation. The transcriptional level of Ca2+-regulating genes was quantified by using qPCR. Both basal intracellular Ca2+ levels and agonist (ConA, OKT3, or thapsigargin)-induced Ca2+ transients were significantly elevated at E3 group (p<0.05 vs. control). However, mitogen-induced cell proliferation was significantly decreased at E3 group (p<0.05 vs. control). In parallel, the transcriptional level of plasma membrane Ca2+-ATPases (PMCA), sarco/endoplasmic reticulum Ca2+-ATPases (SERCA), TRPC1, and P2X7 was significantly downregulated, and the transcriptional level of IP3R2 and RyR2 was significantly upregulated in E3 (p<0.01 vs. control). In summary, this study demonstrated that acute exercise affected intracellular calcium homeostasis, most likely by enhancing transmembrane Ca2+ influx into cells and by reducing expression of Ca2+-ATPases such as PMCA and SERCA. However, altered Ca2+ signals were not transduced into an enhanced T cell proliferation suggesting other pathways to be responsible for the transient exercise-associated immunosuppression.

Raf-like kinases and receptor-like (pseudo)kinase GHR1 are required for stomatal vapor pressure difference response.

Stomatal pores close rapidly in response to low-air-humidity-induced leaf-to-air vapor pressure difference (VPD) increases, thereby reducing excessive water loss. The hydroactive signal-transduction mechanisms mediating high VPD-induced stomatal closure remain largely unknown. The kinetics of stomatal high-VPD responses were investigated by using time-resolved gas-exchange analyses of higher-order mutants in guard-cell signal-transduction branches. We show that the slow-type anion channel SLAC1 plays a relatively more substantial role than the rapid-type anion channel ALMT12/QUAC1 in stomatal VPD signaling. VPD-induced stomatal closure is not affected in mpk12/mpk4GC double mutants that completely disrupt stomatal CO2 signaling, indicating that VPD signaling is independent of the early CO2 signal-transduction pathway. Calcium imaging shows that osmotic stress causes cytoplasmic Ca2+ transients in guard cells. Nevertheless, osca1-2/1.3/2.2/2.3/3.1 Ca2+-permeable channel quintuple, osca1.3/1.7-channel double, cngc5/6-channel double, cngc20-channel single, cngc19/20crispr-channel double, glr3.2/3.3-channel double, cpk-kinase quintuple, cbl1/4/5/8/9 quintuple, and cbl2/3rf double mutants showed wild-type-like stomatal VPD responses. A B3-family Raf-like mitogen-activated protein (MAP)-kinase kinase kinase, M3Kδ5/RAF6, activates the OST1/SnRK2.6 kinase in plant cells. Interestingly, B3 Raf-kinase m3kδ5 and m3kδ1/δ5/δ6/δ7 (raf3/6/5/4) quadruple mutants, but not a 14-gene raf-kinase mutant including osmotic stress-linked B4-family Raf-kinases, exhibited slowed high-VPD responses, suggesting that B3-family Raf-kinases play an important role in stomatal VPD signaling. Moreover, high VPD-induced stomatal closure was impaired in receptor-like pseudokinase GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) mutant alleles. Notably, the classical transient "wrong-way" VPD response was absent in ghr1 mutant alleles. These findings reveal genes and signaling mechanisms in the elusive high VPD-induced stomatal closing response pathway.

Thiosemicarbazone Copper Chelator BLT-1 Blocks Apicomplexan Parasite Replication by Selective Inhibition of Scavenger Receptor B Type 1 (SR-BI)

Coccidian parasites are obligate intracellular pathogens that affect humans and animals. Apicomplexans are defective in de novo synthesis of cholesterol, which is required for membrane biosynthesis and offspring formation. In consequence, cholesterol has to be scavenged from host cells. It is mainly taken up from extracellular sources via LDL particles; however, little is known on the role of HDL and its receptor SR-BI in this process. Here, we studied effects of the SR-BI-specific blocker BLT-1 on the development of different fast (Toxoplasma gondii, Neospora caninum, Besnoitia besnoiti) and slow (Eimeria bovis and Eimeria arloingi) replicating coccidian species. Overall, development of all these parasites was significantly inhibited by BLT-1 treatment indicating a common SR-BI-related key mechanism in the replication process. However, SR-BI gene transcription was not affected by T. gondii, N. caninum and B. besnoiti infections. Interestingly, BLT-1 treatment of infective stages reduced invasive capacities of all fast replicating parasites paralleled by a sustained increase in cytoplasmic Ca++ levels. Moreover, BLT1-mediated blockage of SR-BI led to enhanced host cell lipid droplet abundance and neutral lipid content, thereby confirming the importance of this receptor in general lipid metabolism. Finally, the current data suggest a conserved role of SR-BI for successful coccidian infections.

Arginine-vasopressin mediates counter-regulatory glucagon release and is diminished in type 1 diabetes.

Insulin-induced hypoglycemia is a major treatment barrier in type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (8 to 4 mM) has no significant effect on glucagon secretion in the perfused mouse pancreas or in isolated mouse islets (in vitro), and yet associates with dramatic increases in plasma glucagon. The identity of the systemic factor(s) that elevates circulating glucagon remains unknown. Here, we show that arginine-vasopressin (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Alpha-cells express high levels of the vasopressin 1b receptor (V1bR) gene (Avpr1b). Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of the AVP precursor peptide) and increased blood glucose; effects blocked by pharmacological antagonism of either the glucagon receptor or V1bR. AVP also mediates the stimulatory effects of hypoglycemia produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to insulin-induced hypoglycemia. AVP injection increased cytoplasmic Ca2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. Hypoglycemia also increases circulating levels of AVP/copeptin in humans and this hormone stimulates glucagon secretion from human islets. In patients with T1D, hypoglycemia failed to increase both copeptin and glucagon. These findings suggest that AVP is a physiological systemic regulator of glucagon secretion and that this mechanism becomes impaired in T1D.

Anaesthetic diethyl ether impairs long-distance electrical and jasmonate signaling in Arabidopsis thaliana

General volatile anaesthetics (GVA) inhibit electrical signal propagation in animal neurons. Although plants do not have neurons, they generate and propagate electrical signals systemically from a local damaged leaf to neighbouring leaves. This systemic electrical signal propagation is mediated by ligand-gated glutamate receptor-like (GLR) channels. Here, we investigated the effect of GVA diethyl ether on the systemic electrical and further downstream responses in Arabidopsis thaliana. We monitored electrical signals, cytoplasmic Ca2+ level ([Ca2+]cyt), ultra-weak photon emission, amino acid contents, phytohormone response as well as gene expression in response to heat wounding during diethyl ether anaesthesia. We found complete suppression of electrical and [Ca2+]cyt signal propagation from damaged leaf to neighbouring systemic leaves upon diethyl ether treatment. Concomitantly, jasmonates (JAs) did not accumulate and expression of JA-responsive genes (AOS, OPR3, JAZ10) was not detected in systemic leaves. However local damaged leaves still showed increased [Ca2+]cyt and accumulated high level of JAs and JA-inducible transcripts. An exogenously added GLR ligand, L-glutamate, was not able to trigger Ca2+ wave in etherized plants indicating that GLRs are targeted by diethyl ether, but not specifically. The fact that GVA inhibit electrical signal propagation not only in animals but also in plants is intriguing. However, the cellular response is completely blocked only in systemic leaves; the local damaged leaf still senses damaging stimuli.

Abstract 11475: A Genetically Modified pH Resistant NCX1 Mouse Prevents Calcium Overload During Acidosis and is Cardioprotective

Acidosis inhibits the cardiac sodium(Na)/calcium(Ca) exchanger (NCX1), allegedly contributing to elevated cytoplasmic Na and Ca concentrations during ischemia. Upon reperfusion and restoration of normal pH, a reactivated NCX1 abruptly shifts into reverse (Ca influx) mode because of the abnormally high cellular Na concentration, thus producing massive Ca overload and cell damage. To test the dependence of ischemia/reperfusion (I/R) injury on NCX1’s pH sensitivity, we used CRISPR/Cas9 technology to create mice with a pH-resistant NCX1 by substituting Alanine for Histidine at position 165 (H165A). These mice live into adulthood with slightly reduced LV function (EF% H165A: 50 ± 5; Wildtype (WT): 67 ± 2; p&lt;0.025, N=4-6). In enzymatically isolated adult ventricular myocytes, we used the patch clamp technique and voltage ramps from +80 to -120 mV to confirm that NCX1 current in H165A mice is resistant to pH reduction (from 7.4 to ~pH 6.4 by substituting 80 mM Na-Acetate for equimolar NaCl) when compared to WT animals (% reduction at +80mV: WT 77.7± 2.8, P&lt;0.027 in 3 cells from 2 mice; H165A 0.21 ± 11.35, P=0.31 in 7 cells from 3 mice). We then performed I/R studies in explanted hearts perfused in Langendorff fashion with oxygenated Tyrode’s containing (in mM): 136 NaCl, 1.8 CaCl 2 , 5.4 KCl, 0.33 NaH 2 PO 4 , 1.0 MgCl 2 , 10 HEPES, and 10 glucose, pH 7.4, 37 o C. 20 minutes of stop-flow ischemia were followed by reperfusion for 120 minutes. Necrosis area (pixels 2 ) in WT was 26 ± 3 (N=11), compared to only 9 ± 1 (N=7) in H165A mice (P&lt;0.0004). To compare effects of lower pH on isolated cardiomyocyte Ca, we used a photometric epifluorescence detection system to measure cytosolic Ca in enzymatically isolated ventricular myocytes loaded with the Ca indicator fura-2 AM (2 μM). When pH was lowered to ~6.4, Ca transients (ΔF340/F380) increased in WT from 0.6 to 0.9 (P &lt; 0.0004, N=19 cells from 3 mice) but notably did not change in H165A (0.5 ± 0.03 @ pH 7.4, 0.5 ± 0.04 @ pH 6.4, P=0.307, N=21 cells from 3 mice). pH changes were verified in separate cells using the pH indicator SNARF-AM (10 μM). We conclude that eliminating pH sensitivity in NCX1 has major effects on intracellular Ca regulation, is cardioprotective, and a potential strategy for therapies in patients with prolonged ischemia prior to reperfusion.

Gating of RYR2 channels from the arrhythmic RYR2-P2328S mouse heart and some unexpected actions of flecainide

The P2328S mutation in mice is associated with arrhythmia and spontaneous diastolic calcium release in atrial and ventricular myocytes and there is a corresponding leftward shift in the Ca2+-activation curve for mutant RYR2 channels from homozygous mouse hearts (Salvage et al. 2019. J Cell Sci. https://doi.org/10.1242/jcs.229039). P2328 is located in helical domain 1 (HD1) of RYR2. Local structural changes likely result when structurally active proline residues are replaced by structurally inert serine residues. We speculate that local structural changes in HD1 lead to sequential intradomain and interdomain stearic changes through the protein to the distant channel gate, which favor the open pore conformation. The drug flecainide prevents arrhythmia in humans and mouse models of CPVT by blocking NaV1.5 and RYR2 channels. Conventionally, flecainide blocks RYR2 channels in a voltage-dependent manner. We did not observe voltage-dependent pore block. This was possibly because, in contrast to previous studies, the only channel modulators that we used to produce end-diastolic control channel activity were 1 µM cytoplasmic Ca2+ and 1 mM luminal Ca2+. We observed previously unreported, voltage-independent increases in WT and P2328S channel activity at low flecainide concentrations, followed by a decline in activity at higher concentrations. The increase in activity dominated the effect of flecainide on P2328S channels. These effects suggested high-affinity flecainide binding to an activation site and lower-affinity binding to an inhibition site, both distant from the channel pore (Salvage et al. 2021. Cells. https://doi.org/10.3390/cells10082101). Unlike channel block by flecainide, the drug under our conditions stabilized intrinsic sub-conductance activity at +40 mV and -40 mV. Since flecainide effectively reduces CPVT arrythmia clinically and in animal models, we conclude that voltage-independent inhibition and voltage-dependent channel block prevail under cellular conditions. However, channel activation is important to note as it may be unmasked in other circumstances such as acquired cardiac disorders, mutations, or additional drug applications.

Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp.

Cytosolic Ca2+ plays a key role in plant development. Calcium imaging is the most versatile method to detect dynamic changes in Ca2+ in the cytoplasm. In this study, we obtained viable protoplasts of pulp cells by enzymatic hydrolysis. Isolated protoplasts were incubated with the small-molecule fluorescent reagent (Fluo-4/AM) for 30 min at 37 °C. The fluorescent probes successfully stained cytosolic Ca2+ but did not accumulate in vacuoles. La3+, a Ca2+ channel blocker, decreased cytoplasmic fluorescence intensity. These results suggest that Fluo-4/AM can be used to detect changes in cytosolic Ca2+ in the fruit flesh. In summary, we present a method to effectively isolate protoplasts from flesh cells of the fruit and detect Ca2+ by loading a small-molecule calcium fluorescent reagent in the cytoplasm of pulp cells.

From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control.

Information flow within and between cells depends significantly on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global "fluttering" state. Here, we used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junction proteins. We term these two subpopulations "initiator cells," defined by elevated levels of Phospholipase C (PLC) activity, and "standby cells," which exhibit baseline activity. We found that the type and strength of hormonal stimulation and extent of gap junctional communication jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gαq activity. Our computational model successfully reproduces how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gαq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, we show that perturbations to Ca2+ signaling tune the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states.