Cytosolic Calcium Concentration Research Articles

Cytosolic Sodium Influx in Mesophyll Protoplasts of Arabidopsis thaliana, wt, sos1:1 and nhx1 Differs and Induces Different Calcium Changes.

The sodium influx into the cytosol of mesophyll protoplasts from Arabidopsis thaliana cv. Columbia, wild type, was compared with the influx into sos1-1 and nhx1 genotypes, which lack the Na+/H+ antiporter in the plasma membrane and tonoplast, respectively. Changes in cytosolic sodium and calcium concentrations upon a 100 mM NaCl addition were detected by use of epifluorescence microscopy and the sodium-specific fluorescent dye SBFI, AM, and calcium sensitive Fura 2, AM, respectively. There was a smaller and mainly transient influx of Na+ in the cytosol of the wild type compared with the sos1-1 and nhx1 genotypes, in which the influx lasted for a longer time. Sodium chloride addition to the protoplasts' medium induced a significant increase in cytosolic calcium concentration in the wild type at 1.0 mM external calcium, and to a lesser extent in nhx1, however, it was negligible in the sos1-1 genotype. LiCl inhibited the cytosolic calcium elevation in the wild type. The results suggest that the salt-induced calcium elevation in the cytosol of mesophyll cells depends on an influx from both internal and external stores and occurs in the presence of an intact Na+/H+ antiporter at the plasma membrane. The Arabidopsis SOS1 more effectively regulates sodium homeostasis than NHX1.

Mesenchymal stromal/stem cells: breaking the deadlock in the treatment of multiple organ dysfunction syndrome

Mesenchymal stromal/stem cells: breaking the deadlock in the treatment of multiple organ dysfunction syndrome

Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro: A potential antagonist of corticotropin-releasing factor (CRF)

Teneurin C-terminal associated peptides (TCAP) are natural bioactive peptides that possess anxiety-reducing roles in animals, in vivo, and increase cell viability, in vitro. Although these peptides have some primary structural similarity to corticotropin-releasing factor (CRF), they are derived from the distal extracellular region of the teneurin transmembrane protein where they may act as separate soluble peptides after auto-catalytic cleavage from the teneurin protein following interaction with the cognate teneurin receptor, latrophilin (ADGRL), or expressed as a separate mRNA. However, although the signal transduction mechanism of TCAP in neurons has not been established, previous studies indicate an association with the intracellular calcium flux. Therefore, in this study, we have characterized the TCAP-mediated calcium response in hypothalamic cell lines using single-cell calcium methods with pharmacological antagonists to identify potential calcium channels, in vitro. Under normal circumstances, TCAP-1 reduces cytosolic calcium concentrations by uptake into the mitochondria and efflux through the plasma membrane independently of the teneurins. In doing so, TCAP-1 could inhibit the potential ‘stress’ -inducing actions of CRF.

Similarities and Differences between the Orai1 Variants: Orai1α and Orai1β

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP3 generation, which results in intracellular Ca2+ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca2+ release-activated Ca2+ (CRAC) current and, in association with TRPC1, the store-operated Ca2+ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca2+ channel (ARC/LRC), store-independent Ca2+ influx activated by the secretory pathway Ca2+-ATPase (SPCA2) and the small conductance Ca2+-activated K+ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1β, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1β, the implications of the Ca2+ signals triggered by each variant, and their downstream modulatory effect within the cell.

Hydrogen Sulfide Regulates SERCA2a Ubiquitylation via Muscle RING Finger-1 S-Sulfhydration to Affect Cardiac Contractility in db/db Mice.

Hydrogen sulfide (H2S), as a gasotransmitter, is involved in various pathophysiological processes. Diabetic cardiomyopathy (DCM) is a major complication of diabetes mellitus (DM), which leads to structural and functional abnormalities of the myocardium and eventually causes heart failure (HF). Systolic and diastolic dysfunction are fundamental features of heart failure. SERCA2a, as a key enzyme for calcium transport in the endoplasmic reticulum (ER), affects the process of myocardial relaxation and contraction. H2S can protect the cardiac function against diabetic hearts, however, its mechanisms are unclear. This study found that exogenous H2S affects cellular calcium transport by regulating the H2S/MuRF1/SERCA2a/cardiac contractile pathway. Our results showed that, compared with the db/db mice, exogenous H2S restored the protein expression levels of CSE and SERCA2a, and the activity of SERCA2a, while reducing cytosolic calcium concentrations and MuRF1 expression. We demonstrated that MuRF1 could interact with SERCA2a via co-immunoprecipitation. Using LC-MS/MS protein ubiquitylation analysis, we identified 147 proteins with increased ubiquitination levels, including SERCA2a, in the cardiac tissues of the db/db mice compared with NaHS-treated db/db mice. Our studies further revealed that NaHS administration modified MuRF1 S-sulfhydration and enhanced the activity and expression of SERCA2a. Under hyperglycemia and hyperlipidemia, overexpression of the MuRF1-Cys44 mutant plasmid reduced the S-sulfhydration level of MuRF1 and decreased the ubiquitination level of SERCA2a and the intracellular Ca2+ concentration. These findings suggested that H2S modulates SERCA2a ubiquitination through MuRF1 S-sulfhydration of Cys44 to prevent decreased myocardial contractility due to increased cytosolic calcium.

The Mechanism of Action between Pulsed Radiofrequency and Orthobiologics: Is There a Synergistic Effect?

Radiofrequency energy is a common treatment modality for chronic pain. While there are different forms of radiofrequency-based therapeutics, the common concept is the generation of an electromagnetic field in the applied area, that can result in neuromodulation (pulsed radiofrequency—PRF) or ablation. Our specific focus relates to PRF due to the possibility of modulation that is in accordance with the mechanisms of action of orthobiologics. The proposed mechanism of action of PRF pertaining to pain relief relies on a decrease in pro-inflammatory cytokines, an increase in cytosolic calcium concentration, a general effect on the immune system, and a reduction in the formation of free radical molecules. The primary known properties of orthobiologics constitute the release of growth factors, a stimulus for endogenous repair, analgesia, and improvement of the function of the injured area. In this review, we described the mechanism of action of both treatments and pertinent scientific references to the use of the combination of PRF and orthobiologics. Our hypothesis is a synergic effect with the combination of both techniques which could benefit patients and improve the life quality.

Identification of novel differentially expressed genes in type 1 diabetes mellitus complications using transcriptomic profiling of UAE patients: a multicenter study

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disorder that mainly affects children and young adults. It is associated with debilitating and long-life complications. Therefore, understanding the factors that lead to the onset and development of these complications is crucial. To our knowledge this is the first study that attempts to identify the common differentially expressed genes (DEGs) in T1DM complications using whole transcriptomic profiling in United Arab Emirates (UAE) patients. The present multicenter study was conducted in different hospitals in UAE including University Hospital Sharjah, Dubai Hospital and Rashid Hospital. A total of fifty-eight Emirati participants aged above 18 years and with a BMI < 25 kg/m2 were recruited and forty-five of these participants had a confirmed diagnosis of T1DM. Five groups of complications associated with the latter were identified including hyperlipidemia, neuropathy, ketoacidosis, hypothyroidism and polycystic ovary syndrome (PCOS). A comprehensive whole transcriptomic analysis using NGS was conducted. The outcomes of the study revealed the common DEGs between T1DM without complications and T1DM with different complications. The results revealed seven common candidate DEGs, SPINK9, TRDN, PVRL4, MYO3A, PDLIM1, KIAA1614 and GRP were upregulated in T1DM complications with significant increase in expression of SPINK9 (Fold change: 5.28, 3.79, 5.20, 3.79, 5.20) and MYO3A (Fold change: 4.14, 6.11, 2.60, 4.33, 4.49) in hyperlipidemia, neuropathy, ketoacidosis, hypothyroidism and PCOS, respectively. In addition, functional pathways of ion transport, mineral absorption and cytosolic calcium concentration were involved in regulation of candidate upregulated genes related to neuropathy, ketoacidosis and PCOS, respectively. The findings of this study represent a novel reference warranting further studies to shed light on the causative genetic factors that are involved in the onset and development of T1DM complications.

ПУТИ РЕАЛИЗАЦИИ АПОПТОЗА ЛИМФОЦИТОВ ЧЕЛОВЕКА, ИНДУЦИРОВАННОГО ПЕРОКСИДОМ ВОДОРОДА, И ИХ РЕГУЛИРОВАНИЕ

The changes in marker indicators of apoptotic cell death were studied: the level of functional activity of initiating caspases-8, -9, -12, membrane death receptors Fas, intracellular reactive oxygen species, cytosolic calcium, mitochondrial potential of peripheral blood lymphocytes of donors induced by exposure to hydrogen peroxide at final concentrations of 10-6 and 10-5 mol/l. Activation of initiating caspase-8 associated with membrane death receptors Fas was detected after exposure to hydrogen peroxide lymphocytes at a concentration of 10-6 mol/l. There was a significant increase in the functional activity of initiating caspase-9, the level of Fas receptors and intracellular reactive oxygen species, as well as a decrease in the concentration of cytosolic calcium and the value of the mitochondrial membrane potential 1 and 2 hours after modification of cells with hydrogen peroxide at a concentration of 10-5 mol/l compared with those for intact immunocytes. It was found that apoptosis of lymphocytes under the influence of exogenous hydrogen peroxide is realized with the participation of receptor-mediated caspase, mitochondrial (with activation of caspase-9) and p53-dependent mechanisms. The protective effect of resveratrol (10-6 mol/l) on lymphocytes after exposure to hydrogen peroxide (10-5 mol / l), which is due to its ability to reduce the level of reactive oxygen species in intact and modified hydrogen peroxide immunocytes, as well as deactivate hydrogen peroxide.

Molecular Bases of Serotonin Reuptake Inhibitor Antidepressant-Attributed Effects in COVID-19: A New Insight on the Role of Bradykinins.

Widely available effective drugs to treat coronavirus disease-2019 (COVID-19) are still limited. Various studies suggested the potential contribution of selective serotonin-reuptake inhibitor (SSRI) antidepressants to alleviate the clinical course of COVID-19. Initially, SSRI antidepressant-attributed anti-COVID-19 activity was attributed to their direct agonistic or indirect serotonin-mediated stimulation of sigma-1 receptors (Sig1-R). Thereafter, attention was drawn to the property of SSRI antidepressants to decrease ceramide production, as functional inhibitors of acid sphingomyelinase. Ceramides are cell membrane waxy lipids formed by sphingosine and a fatty acid, playing a major role in receptor signaling and infection. In COVID-19 patients, ceramide production is increased due to acid sphingomyelinase activation. Here, we aimed to review the relationships between bradykinins and the proposed pathways supporting SSRI antidepressant-attributed effectiveness in COVID-19. In COVID-19 patients, bradykinin receptor-B1 stimulation is enhanced following the downregulation of angiotensin-converting enzyme-2, which is responsible for the inactivation of des-Arg9-bradykinin, a bradykinin metabolite, contrasting with the decrease in bradykinin receptor-B2 (BDKRB2) stimulation, which results from the inhibition of cathepsin L, a kininogenase involved in bradykinin production and present at the infection site. Sig1-R stimulation modulates the inflammatory response by regulating cytokine production and counterbalances COVID-19-attributed BDKRB2 inhibition by potentiating its effects on the cytosolic calcium concentration. Moreover, the beneficial effects obtained with acid sphingomyelinase inhibition are parallel to those expected with BDKRB2 stimulation in COVID-19. Altogether, these findings suggest that one ultimate pathway of SSRI antidepressant-attributed anti-COVID-19 activity is the potentiation of BDKRB2 effects shown to be inhibited in COVID-19. In conclusion, SSRI antidepressants are able to interact positively with the pathophysiological mechanisms involved in COVID-19. However, their exact benefits in preventing morbidities or improving the outcome in COVID-19 patients remain unknown.

Induction of cellulase production by Sr2+ in Trichoderma reesei via calcium signaling transduction

Trichoderma reesei RUT-C30 is a well-known high-yielding cellulase-producing fungal strain that converts lignocellulose into cellulosic sugar for resource regeneration. Calcium is a ubiquitous secondary messenger that regulates growth and cellulase production in T. reesei. We serendipitously found that adding Sr2+ to the medium significantly increased cellulase activity in the T. reesei RUT-C30 strain and upregulated the expression of cellulase-related genes. Further studies showed that Sr2+ supplementation increased the cytosolic calcium concentration and activated the calcium-responsive signal transduction pathway of Ca2+–calcineurin-responsive zinc finger transcription factor 1 (CRZ1). Using the plasma membrane Ca2+ channel blocker, LaCl3, we demonstrated that Sr2+ induces cellulase production via the calcium signaling pathway. Supplementation with the corresponding concentrations of Sr2+ also inhibited colony growth. Sr2+ supplementation led to an increase in intracellular reactive oxygen species (ROS) and upregulated the transcriptional levels of intracellular superoxide dismutase (sod1) and catalase (cat1). We further demonstrated that ROS content was detrimental to cellulase production, which was alleviated by the ROS scavenger N-acetyl cysteine (NAC). This study demonstrated for the first time that Sr2+ supplementation stimulates cellulase production and upregulates cellulase genes via the calcium signaling transduction pathway. Sr2+ leads to an increase in intracellular ROS, which is detrimental to cellulase production and can be alleviated by the ROS scavenger NAC. Our results provide insights into the mechanistic study of cellulase synthesis and the discovery of novel inducers of cellulase.Graphical

Vacuolal and Peroxisomal Calcium Ion Transporters in Yeasts and Fungi: Key Role in the Translocation of Intermediates in the Biosynthesis of Fungal Metabolites.

Highlights The intracellular calcium content plays a key role in the expression of genes involved in the biosynthesis and secretion of fungal metabolites.The cytosolic calcium concentration in fungi is maintained by influx through the cell membrane and by release from store organelles.Some MSF transporters, e.g., PenV of Penicillium chrysogenum and CefP of Acremonium chrysogenum belong to the TRP calcium ion channels.A few of the numerous calcium ion transporters existing in organelles of different filamentous fungi have been characterized at the functional and subcellular localization levels.The cytosolic calcium signal seems to be transduced by the calcitonin/calcineurin cascade controlling the expression of many fungal genes. The intracellular calcium content in fungal cells is influenced by a large number of environmental and nutritional factors. Sharp changes in the cytosolic calcium level act as signals that are decoded by the cell gene expression machinery, resulting in several physiological responses, including differentiation and secondary metabolites biosynthesis. Expression of the three penicillin biosynthetic genes is regulated by calcium ions, but there is still little information on the role of this ion in the translocation of penicillin intermediates between different subcellular compartments. Using advanced information on the transport of calcium in organelles in yeast as a model, this article reviews the recent progress on the transport of calcium in vacuoles and peroxisomes and its relation to the translocation of biosynthetic intermediates in filamentous fungi. The Penicillium chrysogenum PenV vacuole transporter and the Acremonium chrysogenum CefP peroxisomal transporter belong to the transient receptor potential (TRP) class CSC of calcium ion channels. The PenV transporter plays an important role in providing precursors for the biosynthesis of the tripeptide δ-(-α-aminoadipyl-L-cysteinyl-D-valine), the first intermediate of penicillin biosynthesis in P. chrysogenum. Similarly, CefP exerts a key function in the conversion of isopenicillin N to penicillin N in peroxisomes of A. chrysogenum. These TRP transporters are different from other TRP ion channels of Giberella zeae that belong to the Yvc1 class of yeast TRPs. Recent advances in filamentous fungi indicate that the cytosolic calcium concentration signal is connected to the calcitonin/calcineurin signal transduction cascade that controls the expression of genes involved in the subcellular translocation of intermediates during fungal metabolite biosynthesis. These advances open new possibilities to enhance the expression of important biosynthetic genes in fungi.

Calmodulin kinase 2 genetically interacts with Rch1p to negatively regulate calcium import into Saccharomyces cerevisiae after extracellular calcium pulse

After a single extracellular 100mM calcium pulse (final concentration), wild type S. cerevisiae exhibits a sharp peak in cytosolic calcium. The concentration drops rapidly in these cells as the calcium is sequestered away in the endoplasmic reticulum, Golgi, and vacuoles leaving resting cytosolic levels higher than their original state, followed by changes in gene expression. In cells lacking calmodulin kinase 2 (Cmk2p),a secondary rise in cytosolic calcium concentration is seen after extracellular calcium increases and the cytosolic calcium is subsequently sequestered. Utilizing double deletions, we demonstrate that Cmk2p is modulating the activity of Rch1p, a known inhibitor of Channel X which is a yeast plasma membrane channel through which calcium ions are transported after an extracellular calcium pulse. We hypothesize that Cmk2p is acting as a regulator of Channel X by activating Rch1p and without CMK2, Channel X inactivation does not occur fully.

Activation of STIM1/Orai1‑mediated SOCE in sepsis‑induced myocardial depression

Unbalanced Ca2+ homeostasis serves an essential role in the occurrence and development of septic myocardial injury. However, the mechanism of Ca2+ homeostasis in septic myocardial depression is poorly understood due to the complexity of Ca2+ transporters in excitable cells. It was therefore hypothesized that cardiac dysfunction, myocardial injury and cardiac apoptosis in septic myocardial depression are associated with elevated intracellular Ca2+ concentrations caused by stromal interaction molecule 1 (STIM1)/Orai calcium release‑activated calcium modulator 1 (Orai1)‑mediated store‑operated Ca2+ entry (SOCE). A septic myocardial depression model was established using the cecal ligation and puncture operation (CLP) in mice and was simulated in H9C2 cells via lipopolysaccharide (LPS) stimulation. Cardiac function, myocardial injury, cardiac apoptosis and the expression levels of Bax, Bcl‑2, STIM1 and Orai1 were quantified invivo at6, 12and24h. Changes in the intracellular Ca2+ concentration, SOCE and the distribution of STIM1 were assessed invitro within 6h. The morphological changes of heart tissue were observed by hematoxylin‑eosin staining. Myocardial cellular apoptosis was determined by TUNEL method. The expression of Bax, Bcl‑2, STIM1 and Orai1 were visualized by western blot. Cytosolic calcium concentration and SOCE were evaluated by confocal microscopy. The results demonstrated that cardiac contractile function was significantly reduced at6h and morphological changes in cardiac tissues, as well as the myocardial apoptosis rate, were markedly increased at6, 12and24h following CLP. mRNA and protein expression levels of Bax/Bcl‑2 were significantly enhanced at6and12h and glycosylation of Orai1 in the myocardium of septic mice was significantly increased at6h following CLP. The intracellular Ca2+ concentration, SOCE, was significantly increased at1‑2h and the clustering and distribution of STIM1 were markedly changed in H9C2 cells at1and2h. These findings suggested that myocardial dysfunction, cardiac injury and myocardial depression may be related to increased intracellular Ca2+ concentration resulting from STIM1/Orai1‑mediated SOCE, which may provide a potential method to alleviate septic myocardial depression.

RuBPY decreases intracellular calcium by decreasing influx and increasing storage.

cis-[Ru(bpy)2(py)NO2](PF6) (RuBPY) is a ruthenium complex nitric oxide (NO) donor that presents a nitrite in its moiety and has been shown to induce vasodilation in various arteries, as well as arterial pressure reduction with no changes in heart rate. Because vascular tone is highly dependent on the cytosolic calcium concentration ([Ca2+ ]c), the current study aimed to investigate the effects of RuBPY on the intracellular mobilization of calcium stores of rat aortic vascular smooth muscle cells. Vascular reactivity experiments were performed in isolated aortic rings that were contracted with a high concentration of KCl or phenylephrine (Phe). Moreover, primary cultured vascular smooth muscle cells were used to measure [Ca2+ ]c by confocal microscopy. The NO donor RuBPY decreased the [Ca2+ ]c and reduced KCl and Phe-induced contractile responses. The selective inhibitor of sarco-endoplasmic Ca-ATPase (SERCA) with thapsigargin impaired the effect of RuBPY on Phe-induced contractile response. RuBPY also reduced caffeine-induced contraction, and the contraction dependent on the capacitive Ca2+ influx. Therefore, our results suggest that NO released from RuBPY decreased [Ca2+ ]c by calcium influx blockade and activation of guanylyl-cyclase-cGMP-GK pathway. These results indicate that RuBPY increases Ca2+ storage in the sarcoplasmic reticulum by SERCA activation and also by capacitive Ca2+ influx inhibition, which is dependent on the intracellular release of nitric oxide from this compound.

Lappaconitine inhibits glutamate release from rat cerebrocortical nerve terminals by suppressing Ca2+ influx and protein kinase A cascade

The inhibition of the excessive release of glutamate in the brain has emerged as a promising new option for developing therapeutic strategies for neurodegenerative disorders. This study investigated the effect and mechanism of lappaconitine, a diterpenoid alkaloid found in species of Aconitum, on glutamate release in rat cerebral cortex nerve terminals (synaptosomes). Here, we report that in the rat cortical synaptosomal preparation, lappaconitine reduced the K+ channel blocker 4-aminopyridine (4-AP)-evoked Ca2+-dependent release of glutamate. The inhibitory effect of lappaconitine on the evoked glutamate release was blocked by the vesicular transporter inhibitor bafilomycin A1 and calcium-chelating agent ethylene glycol tetraacetic acid (EGTA), but was unaffected by exposure to the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate (dl-TBOA). The depolarization-induced elevation of cytosolic calcium concentration ([Ca2+]c) was inhibited by lappaconitine, while the 4-AP-mediated depolarization of the synaptosomal membrane potential was not affected. The inhibition of glutamate release by lappaconitine was markedly decreased in synaptosomes pretreated with the Cav2.3 (R-type) channel blocker SNX-482 or the protein kinase A inhibitor H89. Nevertheless, the lappaconitine-mediated inhibition of glutamate release was not abolished by the intracellular Ca2+-release inhibitors dantrolene and CGP37157. Lappaconitine also significantly decreased the 4-AP-induced phosphorylation of PKA and SNAP-25, a presynaptic substrate for PKA. Our data suggest that lappaconitine reduces Ca2+ influx through R-type Ca2+ channels, subsequently reducing the protein kinase A cascade to inhibit the evoked glutamate release from rat cerebral cortex nerve terminals.

Biocompatibility and immunostimulatory properties of fish collagen and shrimp chitosan towards peripheral blood mononuclear cells (PBMCs)

Several naturally occurring biopolymers are commercially produced from livestock and farmed animals processing wastes, including aquatic organisms. These wastes are considered valuable coproducts of fishery processing industry, from which biopolymers may be recovered and exploited for their bioactive potential. The aim of this work was to prepare polymeric films from collagen and chitosan solutions, extracted from fishery discards, and investigate the cytotoxicity and immunomodulatory activity towards human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy donors and treated with Chitosan, Collagen, Chitosan+Collagen solutions and Chitosan+Collagen film in order to measure the changes in cell viability, cytosolic calcium concentration ([Ca2+]cyt), mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) levels, differentiation and activation of T CD8+ and CD4+ lymphocytes, and cytokine production. Results showed that collagen and chitosan preparations did not show cytotoxic effect, while cellular IL-6, IL-10, and TNF-α release was observed. Chitosan and collagen were able to promote non-cytotoxic PBMCs activation through cytosolic and mitochondrial ROS production. There was a noteworthy phenotyping of lymphocytes T CD8+ and CD4+ counting and an increase of [Ca2+] cyt and ΔΨm levels. These results suggest that chitosan/collagen-based biomaterials produce immunostimulatory effects on PBMC with potential to biomedical approaches.

Alpha-1 Antitrypsin Therapy Modifies Neutrophil Adhesion in Patients with Obstructive Lung Disease.

Alpha-1 antitrypsin deficiency (AATD) is characterized by neutrophil-dominated inflammation resulting in emphysema. The cholesterol-rich neutrophil outer plasma membrane plays a central role in adhesion and subsequent transmigration to underlying tissues. This study aimed to investigate mechanisms of increased neutrophil adhesion in AATD and whether alpha-1 antitrypsin (AAT) augmentation therapy abrogates this effect. Plasma and blood neutrophils were donated by healthy controls (n = 20), AATD (n = 30), and AATD patients after AAT augmentation therapy (n = 6). Neutrophil membrane protein expression was investigated using liquid chromatography-tandem mass spectrometry. The effect of once-weekly intravenous AAT augmentation therapy was assessed by calcium fluorometric, μ-calpain, and cell adhesion assays. Decreased neutrophil plasma membrane cholesterol content (P = 0.03), yet increased abundance of integrin α-M (fold change 1.91), integrin α-L (fold change 3.76), and cytoskeletal adaptor proteins including talin-1 (fold change 4.04) were detected on AATD neutrophil plasma membrane fractions. The described inflammatory induced structural changes were a result of a more than twofold increased cytosolic calcium concentration (P = 0.02), leading to significant calcium-dependent μ-calpain activity (3.5-fold change; P = 0.005), resulting in proteolysis of the membrane cholesterol trafficking protein caveolin-1. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased caveolin-1 and membrane cholesterol content (111.8 ± 15.5 vs. 64.18 ± 7.8 μg/2 × 107 cells before and after treatment, respectively; P = 0.02), with concurrent decreased neutrophil integrin expression and adhesion. Results demonstrate an auxiliary benefit of AAT augmentation therapy, evident by a decrease in circulating inflammation and controlled neutrophil adhesion.

Increased cytosolic calcium buffering contributes to a cellular arrhythmogenic substrate in iPSC-cardiomyocytes from patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life-threatening cardiac arrhythmias. Using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model harbouring a mutation in cardiac troponin T (R173W), we aim to examine the cellular basis of arrhythmogenesis in DCM patients with this mutation. iPSC from control (Ctrl) and DCM-TnT-R173W donors from the same family were differentiated into iPSC-CM and analysed through optical action potential (AP) recordings, simultaneous measurement of cytosolic calcium concentration ([Ca2+]i) and membrane currents and separately assayed using field stimulation to detect the threshold for AP- and [Ca2+]i-alternans development. AP duration was unaltered in TnT-R173W iPSC-CM. Nevertheless, TnT-R173W iPSC-CM showed a strikingly low stimulation threshold for AP- and [Ca2+]i-alternans. Myofilaments are known to play a role as intracellular Ca2+ buffers and here we show increased Ca2+ affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca2+. Similarly, EMD57033, a myofilament Ca2+ sensitiser, replicated the abnormal [Ca2+]i dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca2+ desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca2+ dynamics, normalising Ca2+ transient profile and minimising the occurrence of Ca2+ alternans at physiological frequencies. This finding suggests that increased Ca2+ buffering likely plays a major arrhythmogenic role in patients with DCM, specifically in those with mutations in cardiac troponin T. In addition, we propose that modulation of myofilament Ca2+ sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.

Neuronal Dysfunction Associated with Colorectal Cancer Augments Sensitivity to Chemotherapy-induced Neuropathic Pain

Neuropathic pain is a common and debilitating side effect of many cancer treatments. The platinum-based chemotherapeutic agent oxaliplatin is widely used in treatment regimens for colorectal cancer, but its side effects (including numbness, tingling or sharp, stabbing, 'electrical' pain) are so severe as to become dose limiting in a subset of patients. We set out to determine if the systemic immune system alterations in colon cancer could influence subsequent sensitivity to oxaliplatin therapy. Using two orthotopic mouse models of colon cancer, we perform immunohistochemistry, behavioral analysis, functional calcium imaging, plasma cytokine/chemokine array analysis and mitochondrial functional capacity to determine the extent to which cancer-associated inflammation drives neuronal dysfunction in response to chemotherapy. We found that two distinct colorectal cancer cell lines in two immunocompetent mouse strains induced impaired mitochondrial function in dorsal root ganglion neurons in vitro. This was accompanied by a significant reduction in intra-epidermal nerve fiber density in the plantar hindpaw skin. Multi-electrode array and ratiometric calcium imaging show abnormalities in spontaneous firing and cytosolic calcium concentration in dorsal root ganglion neurons. Finally, we show that tumor-bearing mice show a greater degree of tactile and cold sensitivity in response to a sub-maximal dose of oxaliplatin, despite there being no overt symptoms of neuropathy in tumor-bearing mice that did not receive oxaliplatin. We conclude that colorectal cancer induces 'sub-clinical' neuronal injury that predisposes to neuropathic pain hypersensitivity upon subsequent treatment with oxaliplatin. Future experiments will focus on identifying the soluble factor(s) responsible, and the generalizability of this phenomenon. Grant support from Rita Allen Foundation.

The Sodium-Calcium Exchanger Controls the Membrane Potential of AFT024: A Mesenchymal Stem Cell Hematopoietic Niche Forming Line.

The aim of this study was to characterize the functional expression of sodium-calcium exchangers on AFT024 cell line, a murine model of mesenchymal stem/stromal cells (MSCs) supporting human primitive hematopoiesis. All current-clamp and voltage-clamp experiments were performed using the perforated patch whole-cell recording technique with amphotericin B. The membrane potential of -14 mV shifted to -35 mV when lowering the external sodium concentration to 0.33 mM and an increase of cytosolic calcium concentration was observed. KB-R7943, a selective blocker of cardiac sodium-calcium exchangers, also named NCX1, induced a hyperpolarization at physiological sodium concentration while it blocked the hyperpolarization observed at low sodium concentration. This demonstrates for the first time the presence of the sodium-calcium exchangers in AFT024 cells and provides initial evidence that the membrane potential of these stromal cells is maintained depolarized by this exchanger. Lowering external sodium concentration and KB-R7943 had no effect on the membrane potential of 2018 cells, a nonhematopoietic-supportive cell line. Since NCX1 is differentially expressed in AFT024 cells as compared with nonhematopoietic supportive cells with more restricted differentiation potential, this study suggests a potential role of this sodium-calcium exchanger, in the differentiation process or hematopoietic support of MSCs.