Ca Stores Research Articles

SIRT1 Inhibition Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

Background: SIRT1 is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 increases sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity that is essential to buffer the increase in Ca2+ induced by release from the sarcoplasmic reticulum (SR). Our lab has shown that metabolic syndrome (MetS) impairs SERCA activity in coronary smooth muscle cells and causes coronary artery disease in Ossabaw miniature swine. We hypothesized that SIRT1 inhibition and MetS would impair Ca2+ buffering. 
 Methods: CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT) and mimic the naturally occurring mutation in humans and decrease SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. The cells were exposed to 5 mM caffeine to maximally release stores of Ca2+ from the SR. Ca2+ buffering capacity of each cell was analyzed after the caffeine-induced peak increase to assess Ca2+ efflux and SERCA activity. 
 Results: MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. The rapid phase of Ca2+ buffering due to Ca2+ efflux was not affected by SIRT1 mutation or MetS. The slower phase of Ca2+ buffering due to SERCA activity was impaired only by SIRT1 mutation (p<0.0005), not by MetS. 
 Conclusion: SIRT1 mutation alone inhibited SERCA buffering of Ca2+ in coronary smooth muscle. (Support: NIH T35HL110854, DK120240, DK09751.)

Vascular calcium signalling and ageing.

Changes in cellular Ca2+ levels have major influences on vascular function and blood pressure regulation. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) orchestrate vascular activity in distinct ways, often involving highly specific fluctuations in Ca2+ signalling. Ageing is a major risk factor for cardiovascular diseases, but the impact of ageing per se on vascular Ca2+ signalling has received insufficient attention. We reviewed the literature for age-related changes in Ca2+ signalling in relation to vascular structure and function. Vascular tone dysregulation in several vascular beds has been linked to abnormal expression or activity of SMC voltage-gated Ca2+ channels, Ca2+ -activated K+ channels or TRPC6 channels. Some of these effects were linked to altered caveolae density, microRNA expression or 20-HETE abundance. Intracellular store Ca2+ handling was suppressed in ageing mainly via reduced expression of intracellular Ca2+ release channels, and Ca2+ reuptake or efflux pumps. An increase in mitochondrial Ca2+ uptake, leading to oxidative stress, could also play a role in SMC hypercontractility and structural remodelling in ageing. In ECs, ageing entailed diverse effects on spontaneous and evoked Ca2+ transients, as well as structural changes at the EC-SMC interface. The concerted effects of altered Ca2+ signalling on myogenic tone, endothelium-dependent vasodilatation, and vascular structure are likely to contribute to blood pressure dysregulation and blood flow distribution deficits in critical organs. With the increase in the world's ageing population, future studies should be directed at solving specific ageing-induced Ca2+ signalling deficits to combat the imminent accelerated vascular ageing and increased risk of cardiovascular diseases.

Mechanisms underlying spontaneous phasic contractions and sympathetic control of smooth muscle in the rat caudal epididymis.

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300μm dia.) exhibited SPCs, while distal segments (350-500μm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1μM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1μM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10μM). SPCs were also inhibited by caffeine (1μM), 2-APB (100μM), niflumic acid (100μM), or by lowering extracellular [Cl-] from 134.4 to 12.4mM but not by ryanodine (25μM) or tetracaine (100μM). Electrical field stimulation (EFS) at 2Hz for 60s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.

Epac activation induces an extracellular Ca2+ -independent Ca2+ wave that triggers acrosome reaction in human spermatozoa.

The signaling pathways of the intracellular second messengers cAMP and Ca2+ play a crucial role in numerous physiological processes in human spermatozoa. One such process is the acrosome reaction (AR), which is necessary for spermatozoa to traverse the egg envelope and to expose a fusogenic membrane allowing the egg-sperm fusion. Progesterone and zona pellucida elicit an intracellular Ca2+ increase that is needed for the AR in the mammalian spermatozoa. This increase is mediated by an initial Ca2+ influx but also by a release from intracellular Ca2+ stores. It is known that intracellular Ca2+ stores play a central role in the regulation of [Ca2+ ]i and in the generation of complex Ca2+ signals such as oscillations and waves. In the human spermatozoa, it has been proposed that the cAMP analog and specific agonist of Epac 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (2'-O-Me-cAMP) elicits an intracellular Ca2+ release involved in the AR. To identify the molecular entities involved in the Ca2+ mobilization triggered by 2'-O-Me-cAMP in human spermatozoa. In capacitated human spermatozoa, we monitored Ca2+ dynamics and the occurrence of the AR in real time using Fluo 3-AM and FM4-64 in a Ca2+ -free medium. Epac activation by 2'-O-Me-cAMP induced a Ca2+ wave that started in the midpiece and propagated to the acrosome region. This Ca2+ response was sensitive to rotenone, CGP, xestospongin, NED-19, and thapsigargin, suggesting the participation of different ion transporters (mitochondrial complex I and Na+ /Ca2+ exchanger, inositol 3-phosphate receptors, two-pore channels and internal store Ca2+ -ATPases). Our results suggest that Epac activation promotes a dynamic crosstalk between three different intracellular Ca2+ stores: the mitochondria, the redundant nuclear envelope, and the acrosome. The Ca2+ wave triggered by Epac activation is necessary to induce the AR and to enhance the flagellar beat.

Enhanced Ca2+ handling in thioglycolate-elicited peritoneal macrophages

In macrophage biology, resident peritoneal macrophages (RPMs) and thioglycolate-elicited peritoneal macrophages (TGPMs) have been traditionally utilized as primary cultured models. RPMs and TGPMs exhibit distinct morphological, functional and metabolic characteristics, although it remains unclear how cellular Ca2+ handling differs between them. In our Fura-2 Ca2+ imaging, TGPMs displayed elevated resting Ca2+ levels, increased store Ca2+ contents and facilitated store-operated Ca2+ entry (SOCE) compared with RPMs. The intensified intracellular Ca2+ stores were enriched with major luminal Ca2+-binding proteins inducibly expressed in TGPMs. The elevated resting Ca2+ level was predominantly maintained by constitutive Ca2+ influx, probably through the transient receptor potential (TRP) family members TRPP2, TRPM7 and TRPA1. These TRP family channels seemed to be largely activated in a manner dependent on phospholipase C activity, and together with Orai channels, contributed to SOCE. Moreover, Ca2+-dependent K+ channels efficiently facilitated SOCE by enhancing the Ca2+ driving force in TGPMs. The consolidated cellular Ca2+ handling described may underlie the specialized cell-physiological features of TGPMs, such as vital proliferation, active migration and avid phagocytosis.

STIM1 a calcium sensor promotes the assembly of an ECM that contains Extracellular vesicles and factors that modulate mineralization

Osteoblasts and odontoblasts, are non-excitable cells and facilitate mass calcium transport during matrix mineralization. A sophisticated Ca2+ sensing mechanism is used to maintain Ca2+ homeostasis. STIM1 (Stromal interaction molecule 1) is a calcium sensor protein localized in the ER membrane and maintains calcium homeostasis by initiating the store-operated Ca2+ entry (SOCE) process, following store depletion. The role of STIM1 in dentin mineralization is yet to be elucidated. Therefore, transgenic DPSCs were generated in which overexpression or knockdown of STIM1 was achieved to study its function in matrix mineralization. Gene expression analysis and Alizarin Red staining assay demonstrated upregulation of genes involved in odontogenic differentiation and matrix mineralization with increased calcium deposition with STIM1 overexpression. Topology of the ECM examined by Field Emission Scanning Electron Microscopy (FESEM) showed the presence of large amounts of extracellular microvesicles with mineral deposits. Interestingly, silencing STIM1 resulted in fewer vesicles and less mineral deposits in the ECM. Analysis of the dentin-pulp complex of STIM1- deficient mice by micro-CT show reduced dentin thickness, malformed and highly porous alveolar bone, suggesting a cell intrinsic role for STIM1 in dentin mineralization. Confocal microscopy showed that DMP1-mediated depletion of store Ca2+ resulted in aggregation or “puncta-formation” of STIM1 at the plasma membrane indicative of a gating arrangement with Orai1 for Ca2+ influx. Together, our data provide evidence for an important role for STIM1 in dentin and alveolar bone mineralization by influencing intracellular Ca2+ oscillations that could provide signals for a wide array of cellular functions. Statement of SignificanceCalcium signaling and transport are fundamental to bone and dentin mineralization. Osteoblasts and odontoblasts transport large amounts of Ca2+ to the extracellular matrix. These cells maintain calcium homeostasis by spatially distributed calcium pumps and channels at the plasma membrane. STIM1 an ER Ca2+ sensor protein is an important component of the store-operated calcium entry (SOCE) process. In this study, we examined the role of STIM1 during the differentiation of dental pulp stem cells into functional odontoblasts and formation of mineralized dentin matrix. Stimulation of these cells with DMP1, a key regulatory protein in matrix mineralization, stimulates STIM1-mediated release of ER Ca2+ and SOCE activation. Silencing of STIM1 impairs signaling events, release of exosomes containing matrix proteins and matrix mineralization.

BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation.

Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

Assessing the responses of Sphagnum micro-eukaryotes to climate changes using high throughput sequencing.

Current projections suggest that climate warming will be accompanied by more frequent and severe drought events. Peatlands store ca. one third of the world’s soil organic carbon. Warming and drought may cause peatlands to become carbon sources through stimulation of microbial activity increasing ecosystem respiration, with positive feedback effect on global warming. Micro-eukaryotes play a key role in the carbon cycle through food web interactions and therefore, alterations in their community structure and diversity may affect ecosystem functioning and could reflect these changes. We assessed the diversity and community composition of Sphagnum-associated eukaryotic microorganisms inhabiting peatlands and their response to experimental drought and warming using high throughput sequencing of environmental DNA. Under drier conditions, micro-eukaryotic diversity decreased, the relative abundance of autotrophs increased and that of osmotrophs (including Fungi and Peronosporomycetes) decreased. Furthermore, we identified climate change indicators that could be used as early indicators of change in peatland microbial communities and ecosystem functioning. The changes we observed indicate a shift towards a more “terrestrial” community in response to drought, in line with observed changes in the functioning of the ecosystem.

ORAI2 Down-Regulation Potentiates SOCE and Decreases Aβ42 Accumulation in Human Neuroglioma Cells.

Senile plaques, the hallmarks of Alzheimer’s Disease (AD), are generated by the deposition of amyloid-beta (Aβ), the proteolytic product of amyloid precursor protein (APP), by β and γ-secretase. A large body of evidence points towards a role for Ca2+ imbalances in the pathophysiology of both sporadic and familial forms of AD (FAD). A reduction in store-operated Ca2+ entry (SOCE) is shared by numerous FAD-linked mutations, and SOCE is involved in Aβ accumulation in different model cells. In neurons, both the role and components of SOCE remain quite obscure, whereas in astrocytes, SOCE controls their Ca2+-based excitability and communication to neurons. Glial cells are also directly involved in Aβ production and clearance. Here, we focus on the role of ORAI2, a key SOCE component, in modulating SOCE in the human neuroglioma cell line H4. We show that ORAI2 overexpression reduces both SOCE level and stores Ca2+ content, while ORAI2 downregulation significantly increases SOCE amplitude without affecting store Ca2+ handling. In Aβ-secreting H4-APPswe cells, SOCE inhibition by BTP2 and SOCE augmentation by ORAI2 downregulation respectively increases and decreases Aβ42 accumulation. Based on these findings, we suggest ORAI2 downregulation as a potential tool to rescue defective SOCE in AD, while preventing plaque formation.

Letters from Cuba by Ruth Behar

Reviewed by: Letters from Cuba by Ruth Behar Elizabeth Bush Behar, Ruth Letters from Cuba. Paulsen/Penguin, 2020 [272p] Trade ed. ISBN 9780525516477 $17.99 E-book ed. ISBN 9780525516484 $10.99 Reviewed from digital galleys Ad Gr. 4-7 Papa had been in Cuba for three years, working to bring his family out of political and economic distress in Poland as Nazis consolidate power. With enough saved to send for the first family member, twelve-year-old Esther convinces her father that she, as the eldest, should be chosen over firstborn son Moshe. Papa assents, and the choice proves to be a good one, as Esther's outgoing personality and quickness to pick up a new language are genuine assets to him as he peddles small items in rural areas; even her domestic skills, long criticized by Mama, blossom when she [End Page 464] unleashes her own creativity, designing and sewing dresses that she sells through a Jewish shopkeeper in Havana. It's her interpersonal skill that is arguably the most valuable, as she makes friends ranging from ex-slaves to the town doctor to Chinese merchants, all of whom band together when Nazi sympathizers, emboldened by Hitler's tyranny abroad, mount an anti-immigrant campaign to rid their island of foreigners. Behar finds inspiration for her novel from the experience of her grandmother Esther, a Jewish immigrant who joined her own father in Cuba in 1927. Fictional Esther seems to have stepped out of a decades-old novel as a kind of good-luck magnet who readily vanquishes trouble with wit, charm, and fortunate breaks. Villainous Nazi wannabe Señor Eduardo caves after a one-day strike on his sugar mill; wealthy ladies in a trendy Havana department store can't get enough of Esther's designs; and even Papa opens his mind and heart to neighbors outside his tight-knit circle of Jewish acquaintances. Readers may eyeroll but still find themselves engaged by a World War II refugee story in an unexpected and underexamined setting. An author's note is included. Copyright © 2020 The Board of Trustees of the University of Illinois

Physics of Spontaneous Calcium Oscillations in Cardiac Cells and Their Entrainment.

Mechanical contraction in muscle cells requires Ca to allow myosin binding to actin. Beating cardiomyocytes contain internal Ca stores whose cytoplasmic concentration oscillates. Our theory explains observed single channel dynamics as well as cellular oscillations in spontaneously beating cardiomyocytes. The Ca dependence of channel activity responsible for Ca release includes positive feedback with a delayed response. We use this to predict a dynamical equation for global calcium oscillations with only a few physically relevant parameters. The theory accounts for the observed entrainment of beating to an oscillatory electric or mechanical field.

Persistent cytosolic Ca2+ increase induced by angiotensin II at nanomolar concentrations in acutely dissociated subfornical organ (SFO) neurons of rats

It is known that angiotensin II (AII) is sensed by subfornical organ (SFO) to induce drinking behaviors and autonomic changes. AII at picomolar concentrations have been shown to induce Ca2+ oscillations and increase in the amplitude and frequency of spontaneous Ca2+ oscillations in SFO neurons. The present study was conducted to examine effects of nanomolar concentrations of AII using the Fura-2 Ca2+-imaging technique in acutely dissociated SFO neurons. AII at nanomolar concentrations induced an initial [Ca2+]i peak followed by a persistent [Ca2+]i increase lasting for longer than 1 hour. By contrast, [Ca2+]i responses to 50 mM K+, maximally effective concentrations of glutamate, carbachol, and vasopressin, and AII given at picomolar concentrations returned to the basal level within 20 min. The AII-induced [Ca2+]i increase was blocked by the AT1 antagonist losartan. However, losartan had no effect when added during the persistent phase. The persistent phase was suppressed by extracellular Ca2+ removal, significantly inhibited by blockers of L and P/Q type Ca2+ channels , but unaffected by inhibition of Ca2+ store Ca2+ ATPase. The persistent phase was reversibly suppressed by GABA and inhibited by CaMK and PKC inhibitors. These results suggest that the persistent [Ca2+]i increase evoked by nanomolar concentrations of AII is initiated by AT1 receptor activation and maintained by Ca2+ entry mechanisms in part through L and P/Q type Ca2+ channels, and that CaMK and PKC are involved in this process. The persistent [Ca2+]i increase induced by AII at high pathophysiological levels may have a significant role in altering SFO neuronal functions.

Mechanisms of Calcium Leak from Cardiac Sarcoplasmic Reticulum Revealed by Statistical Mechanics

Heart muscle contraction is normally activated by a synchronized Ca release from sarcoplasmic reticulum (SR), a major intracellular Ca store. However, under abnormal conditions, Ca leaks from the SR, decreasing heart contraction amplitude and increasing risk of life-threatening arrhythmia. The mechanisms and regimes of SR operation generating the abnormal Ca leak remain unclear. Here, we employed both numerical and analytical modeling to get mechanistic insights into the emergent Ca leak phenomenon. Our numerical simulations using a detailed realistic model of the Ca release unit reveal sharp transitions resulting in Ca leak. The emergence of leak is closely mapped mathematically to the Ising model from statistical mechanics. The system steady-state behavior is determined by two aggregate parameters: the analogs of magnetic field (h) and the inverse temperature (β) in the Ising model, for which we have explicit formulas in terms of SR [Ca] and release channel opening and closing rates. The classification of leak regimes takes the shape of a phase β-h diagram, with the regime boundaries occurring at h = 0 and a critical value of β (β∗) that we estimate using a classical Ising model and mean field theory. Our theory predicts that a synchronized Ca leak will occur when h > 0 and β >β∗, and a disordered leak occurs when β <β∗ and h is not too negative. The disorder leak is distinguished from synchronized leak (in long-lasting sparks) by larger Peierls contour lengths, an output parameter reflecting degree of disorder. Thus, in addition to our detailed numerical model approach, we also offer an instantaneous computational tool using analytical formulas of the Ising model for respective ryanodine receptor parameters and SR Ca load that describe and classify phase transitions and leak emergence.

Familial Alzheimer’s disease-linked presenilin mutants and intracellular Ca2+ handling: A single-organelle, FRET-based analysis

An imbalance in Ca2+ homeostasis represents an early event in the pathogenesis of Alzheimer’s disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca2+ content is still debated and the mechanism of action of mutant proteins is unclear.To fulfil the need of a direct investigation of intracellular stores Ca2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca2+ handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca2+ concentration within the main intracellular Ca2+ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) activity. Moreover, by using a cytosolic Ca2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca2+ influx activated by stores depletion (Store-Operated Ca2+ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway.Our data indicate that FAD-linked PSs mutants differentially modulate the Ca2+ content of intracellular stores yet leading to a complex dysregulation of Ca2+ homeostasis, which represents a common disease phenotype of AD.

Amyloid β Oligomers Increase ER-Mitochondria Ca2+ Cross Talk in Young Hippocampal Neurons and Exacerbate Aging-Induced Intracellular Ca2+ Remodeling.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and strongly associated to aging. AD has been related to excess of neurotoxic oligomers of amyloid β peptide (Aβo), loss of intracellular Ca2+ homeostasis and mitochondrial damage. However, the intimate mechanisms underlying the pathology remain obscure. We have reported recently that long-term cultures of rat hippocampal neurons resembling aging neurons are prone to damage induced by Aβ oligomers (Aβo) while short-term cultured cells resembling young neurons are not. In addition, we have also shown that aging neurons display critical changes in intracellular Ca2+ homeostasis including increased Ca2+ store content and Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria. Aging also promotes the partial loss of store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway involved in memory storage. Here, we have addressed whether Aβo treatment influences differentially intracellular Ca2+ homeostasis in young and aged neurons. We found that Aβo exacerbate the remodeling of intracellular Ca2+ induced by aging. Specifically, Aβo exacerbate the loss of SOCE observed in aged neurons. Aβo also exacerbate the increased resting cytosolic Ca2+ concentration, Ca2+ store content and Ca2+ release as well as increased expression of the mitochondrial Ca2+ uniporter (MCU) observed in aging neurons. In contrast, Aβo elicit none of these effects in young neurons. Surprisingly, we found that Aβo increased the Ca2+ transfer from ER to mitochondria in young neurons without having detrimental effects. Consistently, Aβo increased also colocalization of ER and mitochondria in both young and aged neurons. However, in aged neurons, Aβo suppressed Ca2+ transfer from ER to mitochondria, decreased mitochondrial potential, enhanced reactive oxygen species (ROS) generation and promoted apoptosis. These results suggest that modulation of ER—mitochondria coupling in hippocampal neurons may be a novel physiological role of Aβo. However, excess of Aβo in the face of the remodeling of intracellular Ca2+ homeostasis associated to aging may lead to loss of ER—mitochondrial coupling and AD.

Neuroprotection by Regulating the mBDNF/proBDNF Ratio after Cerebral Intracerebral Hemorrhage Is Affected by ATP Administration Through P2X4 r/P38-MAPK/Ca &lt;sup&gt;2+&lt;/sup&gt;/SNAREs Regulation

Background: Brain-derived neurotrophic factor (BDNF) is a neurotrophin that plays a pivotal role in neuronal development by modulating synaptic activity and regeneration. BDNF has two forms, the mature BDNF (mBDNF) and the precursor BDNF (proBDNF), which seem to act in opposite functional ways. The involvement of mBDNF/proBDNF ratio and its role after cerebral intracerebral hemorrhage (ICH) are not fully characterized yet. Methods: To address these issues, adenosine 5′-triphosphate disodium salt hydrate (ATP), a P2 purine agonist that evokes the release of mBDNF, was administrated to regulate mBDNF/proBDNF ratio. Data was evidenced by hematoma volume, neurological scores, edema or blood-brain barrier disruption, HE, Nissl staining and immunofluorescence. To investigate the regulation mechanism of mBDNF/proBDNF ratio by ATP, we used P2X4r LEN, P38-mitogen activated protein kinase (MAPK), Ca2+, and soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) inhibitor. Findings: ATP promoted mBDNF/proBDNF ratio and protected against ICH injury including a reduction in hematoma volume, an increase in neurological scores, alleviation of edema or blood-brain barrier disruption, and an improvement in outcomes of HE, Nissl staining, and immunofluorescence. In addition, the ratio of mBDNF/proBDNF was mediated by P2X4r primarily, and then regulated by ATP-induced activation of P38-MAPK, dependent upon the presence of extracellular and endoplasmic reticulum store Ca2+ and related to SNARE-mediated exocytosis. Interpretation: Our study demonstrates that the mBDNF/proBDNF ratio is able to alleviate ICH injury by ATP administration through P2X4 receptor/P38-MAPK/Ca2+/SNAREs regulation. These results may provide a new strategy to alleviate ICH injury by promoting the mBDNF/proBDNF ratio in neurotrophy. Funding Statement: This work was funded by the National Natural Science Foundation of China (Nos. 81671158 and 81771261), and the Natural Science Youth Foundation of China (No. 81701165), and the Natural Science Foundation of Chongqing Science and Technology Committee, China (No. cstc2015jcyjA10048). Declaration of Interests: The authors declare that they have no competing interests. Ethical Approval Statement: All animals used in this experiment were cared for in strict accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). All animal experimental procedures were approved by the Ethics Committee of Experimental Animals of Chongqing Medical University.

Effects of calpain inhibitor on the apoptosis of hepatic stellate cells induced by calcium ionophore A23187.

We previously showed that changes in calcium concentrations were related to cell apoptosis in vitro. The endoplasmic reticulum (ER) is the main component of calcium storage and signal transduction, and disrupting the balance of intracellular Ca2+ can cause endoplasmic reticulum stress (ERS). In this process, the ER releases stored Ca 2+ into the cytoplasm and activates calpain-2. To further investigate the effect of calpain in hepatic stellate cells (HSCs), in the current study, we examine the effect of N-acetyl-leu-leu-norleucinal (ALLN) on apoptosis resulting from calcium ionophore A23187-induced ERS. Our findings indicate that calpain inhibition reduces calcium ionophore A23187-induced apoptosis of HSCs and decreases the expression of ER stress proteins that may be related to the calpain/caspase signaling pathway.

Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM11–491 and STIM11–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

The early signaling pathway of live yeast cell derivative in THP-1 monocytes

Live yeast cell derivative (LYCD) is a medicinal yeast extract that has been used in the treatment of burns, wounds and hemorrhoids for over 70 years. It has been shown to enhance the closure of skin wounds in diabetic mice by increasing inflammation, angiogenesis, formation of granulation tissue and epithelial migration. An active fraction of LYCD has been identified as a mixture of peptides ranging in size from 5 kDA to 17 kDA. Despite its widespread use over many years, understanding of the mechanism by which LYCD acts to effect tissue repair responses is very limited. In this study, we have used a human monocyte-derived cell line, THP-1, as a representative of the inflammatory component of the wound healing process. We have identified two of the earliest responses to LYCD as an increase in cytoplasmic free calcium ([Ca2+]i) and the transcripts for c-fos. We have found that the increase in [Ca2+]i is both necessary and sufficient to account for the LYCD-induced elevation of c-fos. Furthermore, we have shown that the signaling pathway by which LYCD elevates [Ca2+]i involves both mobilization of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular medium. Mobilization of store Ca2+ occurs first via activation of phospholipase C and this is followed by influx through activation of store operated calcium channels. These results constitute the first delineation of the early steps of the LYCD signaling pathway.

The Role Of P62 On Chronic Oxidative Stress‐Induced Myocytes Ca handling

IntroductionRecent studies have shown that the activity of autophagy impairs, and the accumulation of p62 protein, autophagy substrate, in the heart observes in heart failure patients and mouse models of cardiomyopathy. Accumulations of p62 protein are known to activate the intrinsic cellular stress. Abnormal Ca release is partly responsible for oxidative stress‐induced contractile dysfunction of myocytes in ischemic heart disease. Myocytes Ca release is controlled by a function of sarcoplasmic reticulum (SR) Ca store. However, the role of p62 protein on myocytes Ca regulation in the ischemic heart is not clear.PurposeTo investigate the role of p62 on SR Ca regulation in myocytes isolated from mice with the Isoproterenol‐induced myocardial injury.MethodsTo test our hypothesis, both wild‐type (WT) and p62 knockout (KO) mice were used at 10–12 weeks of age. Myocytes isolated from WT and p62 KO mice (n=3 per group) injected with 200 mg/kg Isoproterenol (ISO; beta agonist, twice per day) for 0 (Base), 5 days, and 10 days. SR Ca handling and contractility measured in field‐stimulated single myocytes loaded with fluorescent Ca indicators (Fura‐2AM; cytosolic Ca loading). Amplitudes of caffeine (10 mM, 5 sec)‐induced Ca transients used as estimates of SR Ca store. Sets of heart from WT and p62 mice were processed for western blot (n=3 per group).ResultsThe p62‐deficient myocytes had no difference of myocytes contractility, and SR Ca handling at the baseline compared to WT myocytes (n=35–45 cells per group). After acute ISO exposure (1μM for 1 min), WT myocytes increased systolic Ca release whereas p62‐deficient myocytes did not. An exposure of a submaximal oxidative stress (1μM; H2O2) in p62‐deficient myocytes, however, increased myocytes contractility and the fraction of Ca released from the SR during each beat (fractional Ca release; systolic Ca release over SR Ca store) after acute ISO stimulation (p&lt;0.05). In WT mice injected with ISO for 5 days and 10 days, myocytes had a biphasic effect on myocytes contractility and fractional Ca release (n=15–45 cells per group, p&lt;0.05). Interestingly, in p62‐deficient myocytes further increased myocytes contractility and fractional Ca release (Figure) on chronic ISO injections from 5 days to 10 days. WT mice hearts with chronic exposure to ISO for 10 days increased p62 protein expressions and suppressed the conversion of LC3 I to LC3 II.ConclusionTaken together, our results suggest that accumulations of p62 protein may directly cause SR Ca defects via beta‐adrenergic signaling in myocytes under chronic myocardial injury.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.