Transient Calcium Increase Research Articles

Gintonin Stimulates Glucose Uptake in Myocytes: Involvement of Calcium and Extracellular Signal-Regulated Kinase Signaling.

Ginseng has anti-hyperglycemic effects. Gintonin, a glycolipoprotein derived from ginseng, also stimulates insulin release from pancreatic beta cells. However, the role of gintonin in glucose metabolism within skeletal muscle is unknown. Here, we showed the effect of gintonin on glucose uptake, glycogen content, glucose transporter (GLUT) 4 expression, and adenosine triphosphate (ATP) content in C2C12 myotubes. Gintonin (3-30 μg/mL) dose-dependently stimulated glucose uptake in myotubes. The expression of GLUT4 on the cell membrane was increased by gintonin treatment. Treatment with 1-3 μg/mL of gintonin increased glycogen content in myotubes, but the content was decreased at 30 μg/mL of gintonin. The ATP content in myotubes increased following treatment with 10-100 μg/mL gintonin. Gintonin transiently elevated intracellular calcium concentrations and increased the phosphorylation of extracellular signal-regulated kinase (ERK). Gintonin-induced transient calcium increases were inhibited by treatment with the lysophosphatidic acid receptor inhibitor Ki16425, the phospholipase C inhibitor U73122, and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Gintonin-stimulated glucose uptake was decreased by treatment with U73122, the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester, and the ERK inhibitor PD98059. These results show that gintonin plays a role in glucose metabolism by increasing glucose uptake through transient calcium increases and ERK signaling pathways. Thus, gintonin may be beneficial for glucose metabolism control.

Stimulatory effect of methylglyoxal on capsaicin-sensitive lung vagal afferents in rats: role of TRPA1.

Methylglyoxal (MG), a reactive metabolic byproduct of glycolysis, is a causative of painful diabetic neuropathy. Patients with diabetes are associated with more frequent severe asthma exacerbation. Stimulation of capsaicin-sensitive lung vagal (CSLV) afferents may contribute to the pathogenesis of hyperreactive airway diseases such as asthma. However, the possibility of the stimulatory effect of MG on CSLV afferents and the underlying mechanisms remain unknown. Our results showed that intravenous injection of MG (25 mg/kg, MG25) in anesthetized, spontaneously breathing rats elicited pulmonary chemoreflexes characterized by apnea, bradycardia, and hypotension. The MG-induced apneic response was reproducible and dose dependent. MG25 no longer evoked these reflex responses after perineural capsaicin treatment of both cervical vagi to block C-fibers' conduction, suggesting that the reflexes were mediated through the stimulation of CSLV afferents. Pretreatment with HC030031 [an antagonist of transient receptor potential ankyrin subtype 1 protein (TRPA1)] or AP18 (another TRPA1 antagonist), but not their vehicle, markedly attenuated the apneic response induced by MG25. Consistently, electrophysiological results showed that pretreatment with HC030031 largely attenuated the intense discharge in CSLV afferents induced by injection of MG25 in open-chest and artificially ventilated rats. In isolated CSLV neurons, the perfusion of MG evoked an abrupt and pronounced increase in calcium transients in a concentration-dependent manner. This stimulatory effect on CSLV neurons was also abolished by HC030031 treatment but not by its vehicle. In conclusion, these results suggest that MG exerts a stimulatory effect on CSLV afferents, inducing pulmonary chemoreflexes, and such stimulation is mediated through the TRPA1 activation.NEW & NOTEWORTHY Methylglyoxal (MG) is implicated in the development of painful diabetic neuropathy. A retrospective cohort study revealed an increased incidence of asthma exacerbations in patients with diabetes. This study demonstrated that elevated circulating MG levels stimulate capsaicin-sensitive lung vagal afferents via activation of TRPA1, which in turn triggers respiratory reflexes. These findings provide new information for understanding the pathogenic mechanism of diabetes-associated hyperreactive airway diseases and potential therapy.

Abstract GS.11: Exercise Therapy During Doxorubicin Treatment Provides Sex-specific Cardioprotection

Moderate-intensity exercise is routinely prescribed to patients with cardiovascular diseases to improve cardiac function. It has also been recommended in patients undergoing anti-cancer chemotherapy to attenuate cardiotoxic effects. We recently identified that moderate endurance exercise in healthy mice causes the remodeling of cardiac physiology in a sex-specific manner. Therefore, we hypothesized that exercise therapy applied during doxorubicin chemotherapy would produce sex-specific cardioprotection. Male and female mice were divided into three groups, 1) sedentary control, 2) DOX - doxorubicin treated at 5 mg/kg/week for 6 weeks, and 3) EXDOX - 2 weeks of forced treadmill running for 2 weeks before DOX + exercise for an additional 6 weeks. Mouse health was tracked daily. Total body weight, echocardiography, and electrocardiography were assessed every 2 weeks. At the end of the protocol, triple parametric optical mapping was performed to determine the remodeling of cardiac metabolism-excitation-contraction coupling (MECC). DOX treatment increased morbidity and reduced total body weight in both males and females. DOX-induced action potential duration (APD) prolongation in both sexes and, additionally, in males alone, increased anisotropic ratio of conduction (index for arrhythmogenicity) and rise time of calcium transients (CaRT). In EXDOX mice, the onset of morbidity was delayed in both males and females compared to DOX mice. Total body weight in male EXDOX mice, but not females, was further reduced compared to DOX treatment alone. Echocardiography revealed increased LV wall thickness in male and female EXDOX mice vs DOX, while stroke volume was reduced in females alone. Next, DOX-induced APD prolongation was prevented in EXDOX females but not males. Furthermore, DOX-induced increase in CaRT was further increased in EXDOX males, in addition to increased calcium transient duration and reduced calcium decay constant. Taken together, these data suggest significant sex dimorphism in cardiac adaptation to DOX treatment and exercise therapy during DOX treatment. Female mice undergoing exercise therapy during DOX treatment demonstrated greater cardioprotection and attenuation of MECC remodeling.

Abaloparatide Maintains Normal Rat Blood Calcium Level in Part Via 1,25-Dihydroxyvitamin D/osteocalcin Signaling Pathway.

The PTH-related peptide(1-34) analog, abaloparatide (ABL), is the second anabolic drug available for the treatment of osteoporosis. Previous research demonstrated that ABL had a potent anabolic effect but caused hypercalcemia at a significantly lower rate. However, the mechanism by which ABL maintains the stability of blood calcium levels remains poorly understood. Our in vivo data showed that ABL treatment (40 µg/kg/day for 7 days) significantly increased rat blood level of 1,25-dihydroxyvitamin D [1,25-(OH)2D] without raising the blood calcium value. ABL also significantly augmented the carboxylated osteocalcin (Gla-Ocn) in the blood and bone that is synthesized by osteoblasts, and increased noncarboxylated Ocn, which is released from the bone matrix to the circulation because of osteoclast activation. The in vitro data showed that ABL (10 nM for 24 hours) had little direct effects on 1,25-(OH)2D synthesis and Gla-Ocn formation in nonrenal cells (rat osteoblast-like cells). However, ABL significantly promoted both 1,25-(OH)2D and Gla-Ocn formation when 25-hydroxyvitamin D, the substrate of 1α-hydroxylase, was added to the cells. Thus, the increased 1,25-(OH)2D levels in rats treated by ABL result in high levels of Gla-Ocn and transient calcium increase in the circulation. Gla-Ocn then mediates calcium ions in the extracellular fluid at bone sites to bind to hydroxyapatite at bone surfaces. This regulation by Gla-Ocn at least, in part, maintains the stability of blood calcium levels during ABL treatment. We conclude that the signaling pathway of ABL/1,25-(OH)2D/Gla-Ocn contributes to calcium homeostasis and may help understand the mechanism of ABL for osteoporosis therapy.

Diadenosine pentaphosphate regulates dendrite growth and number in cultured hippocampal neurons

During the establishment of neuronal circuits, axons and dendrites grow and branch to establish specific synaptic connections. This complex process is highly regulated by positive and negative extracellular cues guiding the axons and dendrites. Our group was pioneer in describing that one of these signals are the extracellular purines. We found that extracellular ATP, through its selective ionotropic P2X7 receptor (P2X7R), negatively regulates axonal growth and branching. Here, we evaluate if other purinergic compounds, such as the diadenosine pentaphosphate (Ap5A), may module the dynamics of dendritic or axonal growth and branching in cultured hippocampal neurons. Our results show that Ap5A negatively modulates the dendrite’s growth and number by inducing transient intracellular calcium increases in the dendrites’ growth cone. Interestingly, phenol red, commonly used as a pH indicator in culture media, also blocks the P2X1 receptors, avoided the negative modulation of Ap5A on dendrites. Subsequent pharmacological studies using a battery of selective P2X1R antagonists confirmed the involvement of this subunit. In agreement with pharmacological studies, P2X1R overexpression caused a similar reduction in dendritic length and number as that induced by Ap5A. This effect was reverted when neurons were co-transfected with the vector expressing the interference RNA for P2X1R. Despite small hairpin RNAs reverting the reduction in the number of dendrites caused by Ap5A, it did not avoid the dendritic length decrease induced by the polyphosphate, suggesting, therefore, the involvement of a heteromeric P2X receptor. Our results are indicating that Ap5A exerts a negative influence on dendritic growth.

Calcium and calcium-related proteins in endometrial cancer: opportunities for pharmacological intervention.

Intracellular calcium ions are key second messengers and play an important role in malignant transformation and cancer progression. Estrogen can evoke intracellular calcium increases through membrane-initiated effects and activate subsequent kinase cascades within minutes in normal and cancerous epithelial cells. Ca2+-related proteins are expressed in normal epithelial cells or endometrial cancer cells, some of which are upregulated by estrogen. Both estrogen-induced transient calcium increases and long-term changes in protein expression levels may be involved in regulating cancer initiation, progression and metastasis. Calcium channel blockers are reported to regulate both the rapid estrogen-induced intracellular Ca2+ increase and cell proliferation, apoptosis and migration, thus having the potential for pharmacological modulators to be repurposed for the treatment of endometrial cancer.

Acute effects of cardiac contractility modulation on human induced pluripotent stem cell-derived cardiomyocytes.

Cardiac contractility modulation (CCM) is an intracardiac therapy whereby nonexcitatory electrical simulations are delivered during the absolute refractory period of the cardiac cycle. We previously evaluated the effects of CCM in isolated adult rabbit ventricular cardiomyocytes and found a transient increase in calcium and contractility. In the present study, we sought to extend these results to human cardiomyocytes using human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) to develop a robust model to evaluate CCM in vitro. HiPSC‐CMs (iCell Cardiomyocytes2, Fujifilm Cellular Dynamic, Inc.) were studied in monolayer format plated on flexible substrate. Contractility, calcium handling, and electrophysiology were evaluated by fluorescence‐ and video‐based analysis (CellOPTIQ, Clyde Biosciences). CCM pulses were applied using an A‐M Systems 4100 pulse generator. Robust hiPSC‐CMs response was observed at 14 V/cm (64 mA) for pacing and 28 V/cm (128 mA, phase amplitude) for CCM. Under these conditions, hiPSC‐CMs displayed enhanced contractile properties including increased contraction amplitude and faster contraction kinetics. Likewise, calcium transient amplitude increased, and calcium kinetics were faster. Furthermore, electrophysiological properties were altered resulting in shortened action potential duration (APD). The observed effects subsided when the CCM stimulation was stopped. CCM‐induced increase in hiPSC‐CMs contractility was significantly more pronounced when extracellular calcium concentration was lowered from 2 mM to 0.5 mM. This study provides a comprehensive characterization of CCM effects on hiPSC‐CMs. These data represent the first study of CCM in hiPSC‐CMs and provide an in vitro model to assess physiologically relevant mechanisms and evaluate safety and effectiveness of future cardiac electrophysiology medical devices.

B-PO03-019 CARDIAC CONTRACTILITY MODULATION STIMULATION DURING SYSTOLE ENHANCES CONTRACTION AND CALCIUM HANDLING PROPERTIES IN HUMAN-INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES

Cardiac contractility modulation (CCM) is a cardiac therapy whereby non-excitatory electrical simulations are delivered to the heart during the absolute refractory period. We previously evaluated effects of CCM and found adult rabbit cardiomyocytes display a transient increase in calcium and contractility. In the present study, we sought to extend these results to human stem-cell cardiomyocytes (hiPSC-CMs). hiPSC-CMs (iCell Cardiomyocytes2, Fujifilm Cellular Dynamic Inc) were studied in monolayer format on Matrigel mattress substrate. Contractility, calcium and electrophysiology were evaluated by image and fluorescence analysis (CellOPTIQ, Clyde Biosciences). Standard clinical CCM pulse parameters were applied with an A-M Systems 4100 pulse generator (Figure 1). Robust CCM response was observed at 11 V/cm (64 mA) for pacing and 22 V/cm (128 mA) for CCM. The first CCM beat displayed a 19.7±0.03 %, (**P<0.01, n=10) contraction amplitude increase that reached steady-state within 5 beats at 8.3±0.16 %. CCM stimulation resulted in faster contraction and relaxation kinetics of 25.9±0.07 % and 18.6±0.07 % respectively. Likewise, calcium amplitude increased by 59.3±0.03% (*P<0.05, n=3). This study provides a comprehensive characterization of the effects of CCM on hiPSC-CMs. These data suggest, CCM exerts its effects by, at a minimum, two calcium centric mechanisms including 1) modulation of L-type calcium channels and 2) increased myofilament calcium sensitivity. These data provide the first study of CCM in hiPSC-CMs and demonstrates the utility of hiPSC-CMs to evaluate safety and effectiveness of cardiac medical devices.

Rapid whole cell imaging reveals a calcium-APPL1-dynein nexus that regulates cohort trafficking of stimulated EGF receptors

The endosomal system provides rich signal processing capabilities for responses elicited by growth factor receptors and their ligands. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors. Activated EGFRs are trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated and degraded. The details of the mechanisms that govern the movements of stimulated EGFRs towards the PNR, are not completely known. Here, exploiting the advantages of lattice light-sheet microscopy, we show that EGFR activation by EGF triggers a transient calcium increase causing a whole-cell level redistribution of Adaptor Protein, Phosphotyrosine Interacting with PH Domain And Leucine Zipper 1 (APPL1) from pre-existing endosomes within one minute, the rebinding of liberated APPL1 directly to EGFR, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within ten minutes. The cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGF receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking and have evolved processes that provide the temporal predictability that typify canonical signaling.

Phosphate Deprivation Can Impair Mechano-Stimulated Cytosolic Free Calcium Elevation in Arabidopsis Roots.

The root tip responds to mechanical stimulation with a transient increase in cytosolic free calcium as a possible second messenger. Although the root tip will grow through a heterogeneous soil nutrient supply, little is known of the consequence of nutrient deprivation for such signalling. Here, the effect of inorganic phosphate deprivation on the root’s mechano-stimulated cytosolic free calcium increase is investigated. Arabidopsis thaliana (cytosolically expressing aequorin as a bioluminescent free calcium reporter) is grown in zero or full phosphate conditions, then roots or root tips are mechanically stimulated. Plants also are grown vertically on a solid medium so their root skewing angle (deviation from vertical) can be determined as an output of mechanical stimulation. Phosphate starvation results in significantly impaired cytosolic free calcium elevation in both root tips and whole excised roots. Phosphate-starved roots sustain a significantly lower root skewing angle than phosphate-replete roots. These results suggest that phosphate starvation causes a dampening of the root mechano-signalling system that could have consequences for growth in hardened, compacted soils.

Abstract 2559: Pharmacological characterization of sigma-2 receptor ligand-induced metabolic stimulation in human SK-N-SH neuroblastoma cells

Abstract We have previously reported a novel metabolically stimulative function of the sigma-2 receptor, with stimulation of glycolytic hallmarks; effects consistent with a pro-survival role and receptor upregulation in cancer cells. These include increased reductive capacity as indicated by stimulation of MTT reduction, increase in cellular ATP level, reduction in basal ROS level, and stabilization of HIF-1α, in human SK-N-SH neuroblastoma cells. The stimulation of MTT reduction was blocked by sigma-2 receptor antagonists, supporting mediation by sigma-2 receptors. Here we further characterize this effect using analogs of the canonical sigma-2 antagonist, SN79. CM764, CM571, and WA504 (sigma-2 Ki = 3.5, 21.7, and 2.5 nM, respectively) all induced dose-dependent stimulation of MTT reduction. At the highest dose examined (30 μM) CM764, CM571, and WA504 induced 45%, 33%, and 75% stimulation of MTT reduction after a 24 h treatment period. Seahorse analysis showed that all three compounds increased both basal and compensatory glycolytic rate as assessed by ECAR. Since these active compounds all have significant sigma-1 receptor affinity (Ki = 8.1-86.6 nM), we examined the effect of structurally similar compounds that lack sigma-1 activity to help rule out its involvement. CM350 (sigma-1 Ki = 1,202 nM; sigma-2 Ki = 83.3 nM) retained stimulative activity, while CM179 (sigma-1 Ki = 1,426 nM; sigma-2 Ki = 2,260 nM) had no activity, suggesting a selective role of the sigma-2 receptor. Our previous study showed that 10 μM CM764 caused a time-dependent increase in HIF-1α level that became prominent at 6 h and continued to increase up to 24 h. HIF-1α is a global regulator of several components of the glycolytic pathway. An examination of the time course revealed that it takes 3 to 6 hours of treatment for ligand-induced increase in MTT reduction to fully develop, consistent with the time course for HIF-1α stabilization. There is evidence that calcium signals can induce expression of HIF-1α. CM764 and CM571 (10 and 30 μM) were found to induce an immediate, transient, and dose-dependent increase in cytosolic calcium in Fura-2 loaded cells. Thapsigargin (150 nM) pretreatment completely eliminated the calcium response, indicating that calcium release derives from endoplasmic reticulum stores. Carbachol also produces a robust thapsigargin-sensitive calcium signal via muscarinic cholinergic receptors, but fails to induce stimulation of MTT reduction. This indicates that if a calcium transient is involved in the mechanism, it must be produced or accompanied by sigma-2 receptor engagement. Taken together, the data suggests that these sigma-2 receptor ligands impact glycolysis via immediate ER calcium release which then induces a latent downstream upregulation of HIF-1α, subsequently resulting in upregulation of glycolytic enzymes and transporters. This will need further investigation to confirm. Citation Format: Bridget M. McVeigh, Alissa K. Oakes, Cheri Z. Liu, Christophe Mesangeau, Walid F. Alsharif, Christopher R. McCurdy, Wayne D. Bowen. Pharmacological characterization of sigma-2 receptor ligand-induced metabolic stimulation in human SK-N-SH neuroblastoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2559.

Abstract 334: Acute Effects of Nonexcitatory Electrical Stimulation During Systole in Human-induced Pluripotent Stem Cell Derived Cardiomyocytes

Cardiac contractility modulation (CCM) is a cardiac therapy whereby non-excitatory electrical simulations are delivered during the absolute refractory period of the cardiac cycle. CCM is indicated for patients with heart failure (Class III/IV) and reduced ejection fraction. We previously evaluated the effects of CCM and found isolated adult rabbit cardiomyocytes (rabbit-CMs) display a transient increase in calcium and contractility. In the present study, we sought to extend these results to human cardiomyocytes using human stem-cell cardiomyocytes (hiPSC-CMs). hiPSC-CMs were studied in both monolayer and isolated cells and results compared to that of rabbit-CMs. Contractility, calcium handling and electrophysiology was evaluated by image and fluorescence analysis. CCM was applied with a custom device covering a range of pulse parameters including standard clinical parameters (Figure 1). Pacing threshold was determined and stimulation performed at twice the diastolic capture threshold. Robust contractile response was observed at 16 V/cm, 70 mA for pacing and 28 V/cm, 120 mA for CCM. Under these conditions hiPSC-CMs and rabbit-CMs displayed increased contraction amplitude of 30 % (1.79 a.u., CCM vs 1.38 a.u., Control) and 22 % (3.67 a.u., CCM vs 3.00 a.u., Control) respectively and faster kinetics that subsided when the CCM signal was eliminated. This study provides a comprehensive characterization of the effects of CCM on hiPSC-CMs and rabbit-CMs in parallel under identical experimental conditions. These data provide the first study of CCM in hiPSC-CMs and demonstrates the potential utility of hiPSC-CMs to evaluate safety and effectiveness of cardiac medical devices.

LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase.

The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

In This Issue

In This Issue

Endothelial Calmodulin and CaMKII Play a Role in Leukocyte Transmigration

Inflammation results in the dilation of blood vessels and recruitment of circulating leukocytes into damaged tissues for repair. A transient increase in endothelial cytosolic free calcium (↑[Ca2+]i) is required for leukocyte transendothelial migration (TEM). We have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the calmodulin‐binding isoleucine‐glutamine (IQ) motif domain is critical for its function during TEM. Calmodulin (CaM) is an intracellular calcium‐binding protein, however, its role in TEM is undescribed. Therefore, we hypothesized that it is involved in the endothelial calcium signaling cascade essential for supporting leukocyte transmigration.In endothelial cells IQGAP1 knockdown reduced TEM substantially. Re‐expressing IQGAP1 truncation mutants containing both the actin‐binding and IQ‐motif domains rescued TEM but truncation mutants lacking the IQ motif domain but did not support TEM.Human endothelial cells treated with the calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Only 13% of leukocytes transmigrated through endothelial monolayers pretreated with 100 mM TFP compared to 84% TEM in control, untreated monolayers (p&lt;0.05). We also looked at the role of calmodulin kinase II (CaMKII). Treating endothelial monolayers with 10 mM KN‐93, a CaMKII inhibitor, substantially reduced leukocyte TEM. Here 22% of leukocytes transmigrated which was significantly reduced compared to control TEM rates (p&lt;0.05). Calmodulin pulldowns also showed that without the IQ‐motif domain IQGAP1 cannot substantially interact with calmodulin further suggesting a link between endothelial calcium, IQGAP1 and CaM.Current experiments focus on inhibiting CaM and CaMKII in endothelial cells using genetically encoded selective inhibitory peptides and inducible endothelial‐specific CaMKII‐deficient mice. Identifying the mechanism by which endothelial Ca2+ influx mediates TEM will provide novel insight into the signaling and also provide potential targets for anti‐inflammatory therapy.Support or Funding InformationR01 HL046849, R01 HL064774, F30 HL134202This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Danger signals activate a putative innate immune system during regeneration in a filamentous fungus.

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

Human Leukocyte Antigen Class I and II Knockout Human Induced Pluripotent Stem Cell-Derived Cells: Universal Donor for Cell Therapy.

BackgroundWe aim to generate a line of “universal donor” human induced pluripotent stem cells (hiPSCs) that are nonimmunogenic and, therefore, can be used to derive cell products suitable for allogeneic transplantation.Methods and ResultshiPSCs carrying knockout mutations for 2 key components (β2 microglobulin and class II major histocompatibility class transactivator) of major histocompatibility complexes I and II (ie, human leukocyte antigen [HLA] I/II knockout hiPSCs) were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene‐editing system and differentiated into cardiomyocytes. Pluripotency‐gene expression and telomerase activity in wild‐type (WT) and HLAI/II knockout hiPSCs, cardiomyocyte marker expression in WT and HLAI/II knockout hiPSC‐derived cardiomyocytes, and assessments of electrophysiological properties (eg, conduction velocity, action‐potential and calcium transient half‐decay times, and calcium transient increase times) in spheroid‐fusions composed of WT and HLAI/II knockout cardiomyocytes, were similar. However, the rates of T‐cell activation before (≈21%) and after (≈24%) exposure to HLAI/II knockout hiPSC‐derived cardiomyocytes were nearly indistinguishable and dramatically lower than after exposure to WT hiPSC‐derived cardiomyocytes (≈75%), and when WT and HLAI/II knockout hiPSC‐derived cardiomyocyte spheroids were cultured with human peripheral blood mononuclear cells, the WT hiPSC‐derived cardiomyocyte spheroids were smaller and displayed contractile irregularities. Finally, expression of HLA‐E and HLA‐F was inhibited in HLAI/II knockout cardiomyocyte spheroids after coculture with human peripheral blood mononuclear cells, although HLA‐G was not inhibited; these results are consistent with the essential role of class II major histocompatibility class transactivator in transcriptional activation of the HLA‐E and HLA‐F genes, but not the HLA‐G gene. Expression of HLA‐G is known to inhibit natural killer cell recognition and killing of cells that lack other HLAs.Conclusions HLAI/II knockout hiPSCs can be differentiated into cardiomyocytes that induce little or no activity in human immune cells and, consequently, are suitable for allogeneic transplantation.

Tranexamic acid blocks the thrombin-mediated delay of epidermal permeability barrier recovery induced by the cedar pollen allergen, Cry j1

We previously demonstrated that Cry j1, the major pollen allergen of Cryptomeria japonica (Japanese cedar), transiently increases protease activity and intracellular Ca2+ concentration in cultured human keratinocytes, and delays recovery after stratum corneum barrier disruption in human skin ex vivo. Topical application of tranexamic acid or trypsin-type serine protease inhibitors accelerates barrier recovery. We hypothesized that tranexamic acid might prevent the transient protease activity increase and the barrier recovery delay induced by Cry j1. Here, we tested this hypothesis and examined the mechanism involved. In cultured human keratinocytes, knock-down of protease-activated receptor 1 (PAR-1) reduced the transient increase of calcium induced by Cry j1, whereas knock-down of PAR-2 did not. Knock-down of thrombin significantly reduced the transient increases of calcium concentration and protease activity. Tranexamic acid, soybean trypsin inhibitor, or bivalirudin (a thrombin inhibitor) also reduced the calcium elevation induced by Cry j1 and/or thrombin. Co-application of tranexamic acid or bivalirudin with Cry j1 to human skin ex vivo blocked the delay of barrier recovery. These results suggest that thrombin and PAR-1 or PAR-1-like receptor might mediate the adverse effects of Cry j1 on human epidermal keratinocytes, and could open up a new strategy for treating inflammatory skin diseases.

A simple end-point assay for calcium channel activity

Cellular calcium signaling events are transient. Hence they are observed in real time using fluorescence imaging or electrophysiological methods that require sophisticated instrumentation and specialized skills. For high throughput assays simple and inexpensive techniques are desirable. Many calcium channels that serve as drug targets have subtypes arising from diverse subunit combinations. These need to be targeted selectively for achieving efficacy and for avoiding side effects in therapies. This in turn increases the number of calcium channels that act as drug targets. We report a novel method for intracellular calcium sensing that utilizes the calcium dependent stable interaction between CaM kinase II (CaMKII) and its ligands such as the NMDA receptor subunit GluN2B. The CaMKII-GluN2B complex formed persists as a memory of the transient increase in calcium. In a cell-based assay system GFP-α-CaMKII expressed in the cytosol responds to calcium by translocating towards GluN2B sequence motif exogenously expressed on mitochondria or endoplasmic reticulum. The resulting punctate fluorescence pattern serves as the signal for intracellular calcium release. The pattern is stable, unaffected by sample processing and is observable without real time imaging. The activities of calcium channel proteins heterologously expressed in HEK-293 cells were detected with specificity using this technique. A calcium sensor vector and a calcium sensor cell line were developed as tools to perform this technique. This technique being simple and less expensive could significantly facilitate high throughput screening in calcium channel drug discovery.

Exploring the Role of Calmodulin and Calcium Signaling in Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. A transient increase in endothelial cytosolic free calcium (Ca2+) is required for leukocyte transendothelial migration (TEM) and transient receptor potential canonical 6 (TRPC6) has been found to mediate this required Ca2+ influx. Furthermore, we have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM. In endothelial cells re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM while constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.Calmodulin (CaM) is a small, highly conserved intracellular calcium‐binding protein, which has been shown to interact with IQGAP1 through the IQ domain. It has also been previously found to regulate TRPC channel activation; however, not much is known regarding its role in TEM. Therefore, we hypothesized that it may be involved in the calcium signaling cascade within endothelial cells essential for supporting leukocyte transmigration.Human endothelial cells treated with calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Endothelial monolayers pretreated with 100 mM TFP for 1 hour drastically reduced leukocyte TEM. Only about 13% of leukocytes were able to transmigrate compared to 84% TEM in control, untreated monolayers (p=0.0025).After identifying the potential role of calmodulin, we generated CaM shRNA to deplete endogenous CaM within endothelial cells, wildtype Flag‐tagged CaM re‐expression constructs and CaM calcium‐binding mutants that we are currently in the process of testing. Identifying the specific mechanism by which endothelial Ca2+ influx through TRPC6 mediates the events of TEM will provide novel insight into the signaling involved in TEM and also provide potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationF30HL134202, R01HL046849, R01HL064774This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.