Dye Fluo-3 Research Articles

CGMP-Dependent Protein Kinase Modulates the Sensitivity of Mesenchymal Stromal Cells to Purinergic Agonists

Calcium signaling induced by ATP in mesenchymal stromal cells (MSCs) from the human adipose tissue was studied by using the Ca2+ dye Fluo-4 and Ca2+ imaging. Previously, purinergic agonists have been found to mobilize Ca2+ in the MSC cytoplasm by stimulating IP3 production and triggering Ca2+ release from Ca2+ stores via IP3 receptors. Here we demonstrated that the phosphodiesterase inhibitor IBMX increased a lag period of ATP-induced Ca2+ responses and also shifted the dose–response curve to higher concentrations. When stimulated by ATP at doses close to the threshold concentration, MSCs became unresponsive in the presence of 50 μM IBMX. PKA inhibitor H89 did not restore MSC responsiveness to ATP lost in the presence of IBMX, while the application of KT-5823, a PKG inhibitor, canceled the inhibitory effect of IBMX. Yet, the cell treatment with KT-5823 resulted in a marked decrease in the ATP response delay. Altogether, the data obtained suggest that PKG modulates the MSC sensitivity to ATP, presumably by phosphorylating IP3 receptors in a cGMP-dependent manner.

Са2+-мобілізація у гладеньких міоцитах сечового міхура щура у відповідь на прикладання капсаїцину

According to the previous studies of the heat-activated TRPV1 ion channel, in the organism it could play not only the canonical role of the nociceptive temperature sensor (activation threshold >42 °С) but also be a polymodal receptor involved in numerous other processes due to the permeability of its selective pore for Ca 2+ ions. Prior studies conducted by other research groups provided indirect evidence obtained with immunochemistry and PCR approaches suggesting the presence of this ion channel in the smooth muscle cells (BSM) of the urinary bladder though the functional data is still absent. In this study we investigated the effect of capsaicin (CAP), a pungent component of hot chili pepper and an agonist of TRPV1, on the transmembrane currents and the Ca 2+ signaling of the bladder smooth muscle cells of the rat. CAP at the saturating concentration of 10 μM had no influence on the transmembrane currents of BSMs registered with the whole-cell patch clamp technique which suggests the absence of TRPV1 in the plasma membrane of these cells. Along with this negative data the control electrophysiological recordings on the dorsal root ganglion (DRG) neurons was conducted showing the presence of CAPS-evoked currents in this cells. On the opposite, the application of this pharmacological agent induced the apparent Ca 2+ response in calcium imaging experiments with the Ca 2+ -sensitive fluorescent dye Fluo-4 AM in 42 % of the BSMs examined. At the same time, the rest of these cells showed no calcium influx though their viability was validated with control cholinoreceptor agonist, carbachol (CCh), in the concentration of 100 μM. Therefore, our findings suggest the previously unreported expression of TRPV1 in the part of the BSMs and its subcellular localization in the sarcoplasmic reticulum of these cells.

Development of a high-throughput fluorescent no-wash sodium influx assay.

Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495–575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1–1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1–1.7 activator veratridine (EC50 10–29 μM) or the NaV1.1–1.8 inhibitor tetracaine (IC50’s 6–66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.

Searching for calcium antagonists for hypertension disease therapy from Moutan Cortex, using bioactivity integrated UHPLC-QTOF-MS.

Calcium channel blockers (CCBs) are currently the most commonly used drugs for the treatment of hypertension. Moutan Cortex (MC), a traditional Chinese herb, has been found to have an anti-hypertensive effect. However, its potential mechanisms in the regulation of intracellular calcium concentration ([Ca2+ ]i ) remain poorly understood. The main objective of this work was to identify the potential calcium antagonists from MC and study their molecular mechanisms. Ultra-high performance liquid chromatography-quadrupole-time-of-fight-mass spectrometry (UHPLC-QTOF-MS) analysis combined with a dual-luciferase reporter assay was utilised to systematically screen the calcium antagonistic active ingredients in the methanol extract of MC. Additionally, the molecular mechanism of these compounds was further studied using live-cell imaging analysis with the calcium ion (Ca2+ ) probe dye fluo-4/AM to monitor changes in [Ca2+ ]i . Three monoterpenoids (paeoniflorin, benzoylpaeoniflorin and mudanpioside C), one phenolic acid (paeonol) and one gallotannin (1,2,3,4,6-O-pentagalloylglucose) were screened out as potential calcium antagonists in MC. Among them, the calcium antagonistic activity of benzoylpaeoniflorin, mudanpioside C and 1,2,3,4,6-O-pentagalloylglucose is first reported. Additionally, paeoniflorin, benzoylpaeoniflorin, mudanpioside C and paeonol can effectively block voltage-operated Ca2+ channels (VOCCs) to exert calcium antagonism, while 1,2,3,4,6-O-pentagalloylglucose plays a role in blocking inositol 1,4,5-trisphosphate receptors (IP3Rs). This work indicated that the anti-hypertensive efficacy of MC acted through multiple components selectively antagonising multiple cell signalling pathways to regulate [Ca2+ ]i . Furthermore, they could be considered as a reference standard for controlling the quality of Chinese medicinal materials.

3127 The effect of common genetic variants in the oxytocin receptor gene on oxytocin response.

OBJECTIVES/SPECIFIC AIMS: Previous studies suggest that genetic variants in the oxytocin receptor (OXTR) may alter oxytocin dose requirement for labor induction and may increase risk for preterm labor and neurodevelopmental disorders. However, the mechanisms of actions of these variants remain unknown. The goal of this study was to functionally characterize common missense and noncoding variants in OXTR. First, we aimed to determine the effects of missense variants on two major aspects of receptor function: calcium signaling and β-arrestin recruitment. Second, we used allelic expression imbalance assays in an effort to identify regulatory single nucleotide polymorphisms (SNPs) in noncoding regions of OXTR that alter OXTR mRNA expression. METHODS/STUDY POPULATION: We used the Exome Aggregation Consortium database to identify the 12 most prevalent missense single nucleotide variants in OXTR. To determine the functional effects of these variants, we transfected human embryonic kidney cells (a common model system used to study receptor function) with wild type OXTR, variant OXTR, or empty vector control. We used the calcium-sensitive dye Fluo4 to quantify intracellular calcium flux in response to oxytocin treatment, and used bioluminescence resonance energy transfer assays to measure recruitment of the signaling partner β-arrestin to the receptor. To investigate potential effects of noncoding SNPs on OXTR mRNA expression, we quantified allele-specific expression of OXTR in human uterine tissue obtained from participants at the time of Cesarean section. We used next-generation sequencing (Illumina MiSeq) to count alleles of a reporter SNP in OXTR exon 3. RESULTS/ANTICIPATED RESULTS: Of the 12 most prevalent missense single nucleotide variants, four were predicted to be deleterious by PolyPhen variant annotation software. We anticipate that these variants will alter receptor signaling through calcium or β-arrestin pathways. We further observed that a reporter SNP in OXTR exon 3 exhibits significant allelic expression imbalance in a subset of our myometrial tissue samples, indicating that OXTR expression may be regulated by a functional SNP. Our current work focuses on discovering the functional SNPs in OXTR responsible for the pattern of allelic expression imbalance seen in mRNA. In the future, we will seek to explore the effects of these variants on uterine function by using genome editing of uterine smooth muscle cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that both missense and noncoding variants may affect OXTR expression and function. Future studies may suggest that OXTR sequencing, genotyping, or expression analysis would be useful to identify individuals likely to respond or fail to respond to safe doses of oxytocin for labor induction. Personalizing approaches for labor induction in this way would increase the safety of oxytocin and potentially reduce maternal morbidity and mortality.

Real-Time Fluorescence Detection of Calcium Efflux During Vacuolar Membrane Fusion.

During in vitro homotypic yeast vacuole fusion Ca2+ is transported into and out of the organelle lumen. In vitro, Ca2+ is taken up from the medium by vacuoles upon the addition of ATP. During the docking stage of vacuole fusion Ca2+ is effluxed from the lumen upon the formation of trans-SNARE complexes between vesicles. Here we describe a real-time fluorescence-based assay to monitor the transport of this cation using purified organelles. Extraluminal Ca2+ is detected when the cation binds the low-affinity fluorescent dye Fluo-4 dextran. This allows for the use of a 96-well microtiter plate to be read in a fluorescence plate reader. Thus, in addition to a curve of calibrated Ca2+ standards, up to 91 experimental conditions can be monitored in a single microplate using this method.

Calcium movement in smooth muscle and evaluation of graded functional intercellular coupling.

Spontaneous activity of vascular smooth muscle is present in small arteries and some venous tissues like the hepatic portal vein. Whereas the ability to generate rhythmic membrane potential changes is expressed in a high number of primary oscillators, the generation of physiological tone and phasic activity requires synchronization of specialized pacemaker activity (Interstitial Cajal-like cells) by intercellular propagation and regeneration of excitation or a strong coupling mechanism of smooth muscle cells. The aim of this study was to deduce oscillator coupling by analyzing the spatiotemporal homogeneity of calcium oscillations within a native tissue preparation. Portal vein tissue was loaded with a calcium-sensitive dye (Fluo-3). By combining confocal microscopy and computation of spatial auto- and cross-correlation of the calcium signals, temporal and spatial coupling between cells was characterized. Spontaneous oscillations of calcium signals were measured at different predefined regions of interest. Cross-correlation analysis of these signals revealed that their damping was very similar in all directions of the investigated z-plane. In single experiments, improved cell-to-cell coupling was seen when noradrenaline (1-10 μM) was added to the bath-solution. With the chosen parameters of frame refresh, the velocity of signal propagation was faster than the maximum detectable velocity, but it could be estimated to exceed 0.1 mm/s. Correlative Network Analysis is a new and very useful tool to determine the functional coupling parameters of quasi-homogenous biological networks and their temporal changes. The action and significance of pharmacological modulators can be well studied on cellular and functional aspects with this newly introduced technique in biological sciences.

Mechanism of 2-aminoethoxydiphenylboronic acid in acetylcholine-inducing Ca2+ signals in acute dissociated mouse pancreatic acinar cells

Objective To reveal the mechanism of 2-aminoethoxydiphenyl borate (2APB) modulates intracellular Ca2+ oscillations. Methods Intracellular Ca2+ signals was monitored directly using Ca2+ -dimensional dye (fluo-4) imaging technique in acute dissociated mouse pancreatic acinar cells. The effect and mechanism of 2APB on acetylcholine (Ach)-induced Ca2+ oscillations were examined by patch-clamp whole cell recording. Results (1) 2APB-induced modulations in acinar cell Ca2+ signals were dependent on 2APB concentrations, with increase of 2APB concentrations (1-100 μmol/L), the amplitude and width of Ca2+ oscillations were increased. (2) 2APB enhanced ACh-induced Ca2+ oscillations were also dependent on the concentrations of ACh. When ACh was less than 5 nmol/L, 2APB did not show effect, between ACh 10-30 nmol/L, 2APB potentiated ACh-induced oscillations, while ACh was higher than 100 nmol/L, 2APB inhibited Ca2+ responses. (3) 2APB enhanced ACh-induced Ca2+ oscillations through extracellular rather than intracellular targets because no effect was found by intracellular application of 100 μmol/L 2APB on 10 nmol/L ACh-induced Ca2+ oscillations (P=0.378, n=6); (4) 2APB significantly blocked SOCE, which can reduced the 3 μmol/L TG-induced Ca2+ oscillations current to (3.0±4.8)% (P=0.000, n=6), suggesting that SOCE may be the target of 2APB to enhance Ca2+ oscillations. (5) SOCE blocker GSK can mimic 2APB’s effect to enhance Ca2+ oscillations (P=0.000, n=7). (6) Using thapsigargin to empty Ca2+ pools, the Ca2+ oscillations current become (93.0±9.5)% (P=0.427, n=7) when 2APB added. In other words, 2APB’s effect was eliminated, suggesting that 2APB-induced Ca2+ oscillations is dependent on Ca2+ pool’s conditions. Conclusion At low ACh concentrations (10-30 nmol/L) (where Ca2+ pool is partially emptied and SOCE is open), 2APB blocks SOCC and promotes Ca2+ further release from Ca2+ pool, resulting in Ca2+ oscillation enhancement. Wherease at higher ACh concentrations (>100 nmol/L), 2APB blocks SOCC and prevents Ca2+ pool refilling from extracellular Ca2+ , thus reduces Ca2+ signal response. Key words: 2-aminoethoxydiphenyl borate; Calcium signals; Pancreatic acinar cells; Patch clam

Only a subpopulation of mouse sperm displays a rapid increase in intracellular calcium during capacitation.

Mammalian sperm must undergo a functionally defined process called capacitation to be able to fertilize oocytes. They become capacitated in vivo by interacting with the female reproductive tract or in vitro in a defined capacitation medium that contains bovine serum albumin, calcium (Ca2+ ), and bicarbonate (HCO3- ). In this work, sperm were double stained with propidium iodide and the Ca2+ dye Fluo-4 AM and analyzed by flow cytometry to determine changes in intracellular Ca2+ concentration ([Ca2+ ]i ) in individual live sperm. An increase in [Ca2+ ]i was observed in a subpopulation of capacitated live sperm when compared with noncapacitated ones. Sperm exposed to the capacitating medium displayed a rapid increase in [Ca2+ ]i within 1 min of incubation, which remained sustained for 90 min. These rise in [Ca2+ ]i after 90 min of incubation in the capacitating medium was evidenced by an increase in the normalized median fluorescence intensity. This increase was dependent on the presence of extracellular Ca2+ and, at least in part, reflected the contribution of a new subpopulation of sperm with higher [Ca2+ ]i . In addition, it was determined that the capacitation-associated [Ca2+ ]i increase was dependent of CatSper channels, as sperm derived from CatSper knockout (CatSper KO) or incubated in the presence of CatSper inhibitors failed to increase [Ca2+ ]i . Surprisingly, a minimum increase in [Ca2+ ]i was also observed in CatSper KO sperm suggesting the existence of other Ca2+ transport systems. Altogether, these results indicate that a subpopulation of sperm increases [Ca2+ ]i very rapidly during capacitation mainly due to a CatSper-mediated influx of extracellular Ca2+ .

Nanosecond pulsed electric field induces calcium mobilization in osteoblasts

Nanosecond pulsed electric field (nsPEF) has the ability to induce a host of intracellular biochemical processes in living cells as a function of parameter setting. In vitro experiments proved that nsPEF stimulation could remarkably promote biomineralization processes and new bone formation. However, the impact of nsPEF parameter settings on the calcium flux of osteoblasts, as well as the intracellular mechanisms underlying those observations have yet to be elucidated. In this study, live osteoblast-like MG63 cells were loaded with fluorescence indicator dye fluo-4 AM. nsPEF stimulation could induce intracellular calcium mobilization in MG63 cells with no refractory period. We confirmed that moderate output voltage (4–8 KV) and large pulse number (25) were required for efficient nsPEF irritation of MG63 cells. Additionally, nsPEF stimulation-induced calcium flux in MG63 cells was dramatically reduced with the treatment by ethylene glycol tetraacetic acid (EGTA), proving that the intake of extracellular calcium ions is crucial for increasing intracellular calcium concentration of nsPEF-treated MG63 cells. Our preliminary study investigated the mechanisms underlying nsPEF stimulation-induced calcium mobilization in osteoblast-like cells, and it has the potential to accelerate the application of nsPEF stimulation in new bone formation.

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.

Bubbles Induce Endothelial Microparticle Formation via a Calcium-Dependent Pathway Involving Flippase Inactivation and Rho Kinase Activation

Background/Aims: Intravascular bubbles can exert pleiotropic detrimental effects, partly by inducing endothelial microparticles (EMPs) production, which play critical roles in cell communication and vascular inflammation cascades. However, the underlying mechanisms remain unclear. This study aimed to delineate the possible mechanisms involving bubble-induced EMPs formation. Methods: Human umbilical vein endothelial cells (HUVECs) were contacted by bubbles and EMPs level in supernatant were quantified by flow cytometry. Cytoplasmic calcium (Ca²⁺) was measured by the Ca²⁺ binding dyes Fluo-3 AM and flippase activity was assessed by translocation rate of fluorescent phosphatidylserine (PS) analogue NBD-PS. Protein levels of phospho-myosin light chain (MLC, a Rho kinase substrate) and phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were determined by western blotting. The score of actin colocalization was assessed by phalloidin-FITC using an immunofluorescent microscopy. Results: EMPs level markedly increased after bubble stimulus. Cytoplasmic calcium (Ca²⁺) significantly elevated (P< 0.05), flippase activity decreased (P< 0.05), protein levels of phospho- MLC and phospho- ERK1/2 significantly increased (P< 0.05, P < 0.05), and the score of actin colocalization markedly reduced (P< 0.05) in bubble-stimulated HUVECs. All the above changes except the increase in phospho-ERK1/2 can be reversed by Ca²⁺ channel blocker LaCl₃ (P< 0.05). Additionally, MLC phosphorylation was significantly inhibited and actin colocalization markedly increased by Rho kinase inhibitor pretreatment and more importantly, bubble-induced EMPs markedly decreased. Conclusions: These results demonstrate that bubble stimulates EMPs formation by cytoplasmic Ca²⁺ elevation and subsequently activating Rho kinase pathway and cytoskeleton reorganization. Simultaneously, cytoplasmic Ca²⁺ inhibits the flippase activity and subsequently increases phosphatidylserine exposure, which also contributes to EMPs formation.

Calcium release from intracellular stores is involved in mitochondria depolarization after lowering extracellular pH in rat brain synaptosomes

In the brain, pH can be lowered in both healthy and disease states. Previously, we showed that moderate extracellular acidification (down to pHo 7.0), but not intracellular acidification, leads to mitochondrial depolarization in synaptosomes. This indicates that the plasma membranes of neuronal presynaptic endings have proton receptors that can induce mitochondrial dysfunction when activated. In the present paper we attempt to identify this hypothetical receptor. First, we have demonstrated that lowering pHo to 7.0 does not induce sodium influx as monitored by the fluorescent dye Sodium Green. This fact, in conjunction with the absence of calcium influx in the same conditions - demonstrated previously, excludes ion channels as possible receptors. However, we showed that acidification-induced mitochondrial depolarization is sensitive to thapsigargin - an inhibitor of calcium release from intracellular stores, U73122 - an inhibitor of phospholipase C, as well as Cu2+ and Zn2+, which can block the metabotropic proton receptor ovarian cancer G protein-coupled receptor 1 (OGR1). Furthermore, using fluorescent dye Fluo-3 we have demonstrated that moderate extracellular acidification induces a cytosolic calcium increase. Excess calcium was scavenged by mitochondria (monitored by fluorescent dye Rhod-2). Our results suggest that the metabotropic OGR1 is a hypothetical presynaptic receptor for low pH. Its activation leads to phospholipase C activation and calcium release from the endoplasmic reticulum followed by accumulation in mitochondria, which likely causes a decrease in mitochondrial membrane potential.

Effects of SCN5A mutation on intracellular calcium concentration

Objective: To construct the delQKP1507-1509 mutant of SCN5A channel, and to study effects of this mutation on intracellular calcium concentration. Methods: SCN5A delQKP1507-1509 mutation was engineered using site-directed mutagenesis. Wild-type (WT), mutant (MT) and mixed (the mixture of wild-type and mutant plasmids at a ratio of 1:1) Na v 1.5 plasmids were transfected into human embryonic kidney 293 (HEK293) cells respectively. Intracellular free calcium concentration were determined by laser confocal scanning microscope (LCSM) after the cells loaded with the Ca 2+ indicator dye Fluo-3/AM. Results: Gel electrophoresis and DNA direct sequencing proved a deletion of nine base pairs in the SCN5A gene at position 1507-1509. Protein expression and localization of wild-type and mutant SCN5A gene in HEK293 cells were detected under the LSCM. Fluorescence intensity in wild-type group, mutant group and co-expression group (1:1 expression of WT and MT sodium channel) mimicking the heterozygous state were 79.3683±3.051, 94.6165±8.383, 90.7463±6.421 respectively, and there was no significant difference among the three groups (P>0.05). Conclusions: SCN5A delQKP1507-1509 mutant is successfully constructed and transfected into HEK293 cells. The mutation does not affect calcium concentration and further study is required to describe the possible pathophysiological mechanisms of dilated cardiomyopathy phenotype in sodium channel overlap syndromes caused by this SCN5A delQKP1507-1509 mutation.

Calcium Imaging of Nerve-Mast Cell Signaling in the Human Intestine.

Introduction: It is suggested that an altered microenvironment in the gut wall alters communication along a mast cell nerve axis. We aimed to record for the first time signaling between mast cells and neurons in intact human submucous preparations.Methods: We used the Ca2+ sensitive dye Fluo-4 AM to simultaneously image changes in intracellular calcium [Ca+2]i (%ΔF/F) in neurons and mast cells. Data are presented as median with interquartile ranges (25/75%).Results: We recorded nerve responses in 29 samples upon selective activation of 223 mast cells by IgE receptor cross linking with the antibody mAb22E7. Mast cells responded to mAb22E7 with a median [Ca+2]i increase of 20% (11/39) peaking 90 s (64/144) after the application. Only very few neurons responded and the median percentage of responding neuronal area was 0% (0/5.9). Mast cell activation remained in the presence of the fast sodium channel blocker tetrodotoxin. Specific neuronal activation by transmural electrical field stimulation (EFS) in 34 samples evoked instantaneously [Ca+2]i signals in submucous neurons. This was followed by a [Ca+2]i peak response of 8%ΔF/F (4/15) in 33% of 168 mast cells in the field of view. The mast cell response was abolished by the nerve blocker tetrododoxin, reduced by the Calcitonin Gene-Related Peptide receptor 1 antagonist BIBN-4096 and the Vasoactive Intestinal Peptide receptor antagonist PG97-269, but not by blockade of the neurokinin receptors 1–3.Conclusion: The findings revealed bidirectional signaling between mast cells and submucous neurons in human gut. In our macroscopically normal preparations a nerve to mast cell signaling was very prominent whereas a mast cell to nerve signaling was rather rare.

Simultaneous Measurement of Contraction and Calcium Transients in Stem Cell Derived Cardiomyocytes

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) provide a powerful platform for disease modeling and drug development in vitro. Traditionally, electrophysiological methods or fluorescent dyes (e.g. calcium) have been used in their functional characterization. Recently, video microscopy has enabled non-invasive analysis of CM contractile motion. Simultaneous assessments of motion and calcium transients have not been generally conducted, as motion detection methods are affected by changing pixel intensities in calcium imaging. Here, we present for the first time a protocol for simultaneous video-based measurement of contraction and calcium with fluorescent dye Fluo-4 videos without corrections, providing data on both ionic and mechanic activity. The method and its accuracy are assessed by measuring the effect of fluorescence and background light on transient widths and contraction velocity amplitudes. We demonstrate the method by showing the contraction-calcium relation and measuring the transient time intervals in catecholaminergic polymorphic ventricular tachycardia patient specific iPSC-CMs and healthy controls. Our validation shows that the simultaneous method provides comparable data to combined individual measurements, providing a new tool for measuring CM biomechanics and calcium simultaneously. Our results with calcium sensitive dyes suggest the method could be expanded to use with other fluorescent reporters as well.

Oxysterol, 5α-cholestan-3-one, modulates a contractile response to β2-adrenoceptor stimulation in the mouse atria: Involvement of NO signaling

AimAtrial β2-adrenoceptors provide an important mechanism for regulation of cardiac function and changes in their downstream signaling are involved in processes underlying heart disorders. We have investigated the mechanism by which the cholesterol metabolite 5α-cholestan-3-one (5ɑCh3) modulates inotropic effect of β2-adrenoceptor agonist fenoterol. Main methodsAtria from mice were electrically stimulated and changes in contraction amplitude in response to fenoterol were studied in 5ɑCh3-pretreated samples. Intracellular Ca2+ and NO levels were estimated using fluorescent dyes Fluo-4 and DAF-FM, respectively. Key findingsBy itself 5αCh3 that appears in the circulation under some pathological conditions had a negligible influence on contraction, Ca2+-transient and NO production. However, pretreatment with 5αCh3 markedly attenuated the positive inotropic effect of fenoterol which was accompanied by an increase in the NO synthesis. Unexpectedly, the oxysterol also augmented an enhancement of Ca2+-transient amplitude in response to fenoterol. Under conditions of a pharmacological inhibition of Gi-protein/Akt/NO synthase/protein kinase G signaling, 5αCh3 augmented the inotropic effect of fenoterol. Herein, Akt antagonist suppressed the increase in NO production, while inhibition of NO synthesis did not modify the increased amplitude of the Ca2+-transient. Along similar lines, enrichment of plasma membranes with cholesterol reduced the stimulatory effect of 5αCh3 on β2-adrenoceptor-evoked NO production, but not on the Ca2+-transient amplitude, leading to an elevation of the positive inotropic response to fenoterol. SignificanceThese data suggest that 5ɑCh3 potentiates the effect of pharmacological β2-adrenoceptor activation on both NO production and Ca2+ transient via independent mechanisms, thereby affecting the positive inotropy.

Abstract 71: Ca 2+ Reuptake is Reduced in the Absence of Insulin Receptors in Mouse Cardiomyocytes

Left ventricle diastolic dysfunction (LVDD), characterized by slow or incomplete relaxation of the ventricle during diastole, is an important contributor to heart failure development in diabetic patients. LVDD can be found in pre-diabetic patients as well as in almost half of asymptomatic patients with well-controlled diabetes. Reduced Ca 2+ reuptake into the sarcoplasmic reticulum (SR) by the SR Ca 2+ ATPase (SERCA) is an important contributor to the slow relaxation observed in diastolic dysfunction. Despite this there are still conflicting results with respect to SERCA expression and function in animal models of diabetes, due perhaps to differences in the duration and severity of diabetes and the presence of systemic complications that can per se alter SERCA expression and/or function. Here, we tested the hypothesis that the impaired relaxation observed in diabetic mice is a consequence of the direct effect of insulin on SERCA. We measured Ca 2+ transient relaxation in isolated cardiomyocytes from 8 weeks cardiomyocyte-selective insulin receptor knockout (CIRKO) mice (Cre-IR lox / lox ), , which lack systemic complications, and compared them with their WT littermates (IR lox / lox ) . Cardiomyocytes loaded with the Ca 2+ dye Fluo-4 were field stimulated at 2 and 3.3 Hz, at 37°C. The intracellular Ca 2+ was monitored using a custom-made epifluorescence system. SERCA and Na + /Ca 2+ exchanger (NCX) expression level was reported as the fold change relative to WT. We found that Ca 2+ transient relaxation was prolonged at both frequencies in CIRKO compared with WT, while retaining a normal frequency-dependent acceleration of relaxation. The exponential time constants were 104±4 (WT) vs. 190±16 (CK) ms at 3.3 Hz, n= 20/group, p&lt;0.001 and 111±6 (WT) vs. 226±19 (CK) ms at 2 Hz, n= 20/group, p&lt;0.001). We also found a reduction in SERCA expression (mRNA: 0.65±0.03, n=10, p&lt;0.001; protein:0.62±0.11, n=4, p&lt;0.05) with no difference in NCX expression (mRNA: 0.92±0.06 n=10/group, p=0.5; protein 0.96±0.05, n=6/group, p=0.2). In conclusion, our findings suggest that the slower Ca 2+ removal observed in the systemic models of diabetes is a direct effect of insulin on SERCA, which could contribute to the LVDD observed in diabetic patients.

Tryptase potentiates enteric nerve activation by histamine and serotonin: Relevance for the effects of mucosal biopsy supernatants from irritable bowel syndrome patients.

We previously showed that mucosal biopsy supernatants from irritable bowel syndrome patients activated neurons despite low concentrations of tryptase, histamine, and serotonin which individually would not cause spike discharge. We studied the potentiating responses between these mediators on excitability of enteric neurons. Calcium-imaging was performed using the calcium-sensitive dye Fluo-4 AM in human submucous plexus preparations from 45 individuals. Histamine, serotonin, and tryptase were applied alone and in combinations to evaluate nerve activation which was assessed by analyzing increase in intracellular Ca2+ ([Ca2+ ]i ), the proportion of responding neurons and the product of both defined as Ca-neuroindex (NI). Protease activated receptor (PAR) 2 activating peptide, PAR2 antagonist and the serine protease-inhibitor FUT-175 were used to particularly investigate the role of proteases. Histamine or serotonin (1μmol/L each) evoked only few small responses (median NI [25%/75%]: 0 [0/148]; 85 [0/705] respectively). Their combined application evoked statistically similar responses (216 [21/651]). Addition of the PAR2 activator tryptase induced a significantly higher Ca-NI (1401 [867/4075]) compared to individual application of tryptase or to coapplied histamine and serotonin. This synergistic potentiation was neither mimicked by PAR2 activating peptide nor reversed by the PAR2 antagonist GB83, but abolished by FUT-175. We observed synergistic potentiation between histamine, serotonin, and tryptase in enteric neurons, which is mediated by proteolytic activity rather than PAR2 activation. This explained neuronal activation by a cocktail of these mediators despite their low concentrations and despite a relatively small PAR2-mediated response in human submucous neurons.

In vivo imaging of Ca2+ accumulation during cotton fiber initiation using fluorescent indicator YC3.60.

Non-tip-focused Ca 2+ gradient indicated by genetically expressing a FRET-based calcium sensor YC3.60 was established in spherical expanding cotton fibers, which is vital for cotton fiber initiation. Cotton fiber is a single cell elongated from ovule epidermis. It is not only the most important natural fiber used in the textile industry but also an ideal model for studying cell differentiation and elongation. Before linear cell growth, cotton fibers undergo spherical expansion at the beginning of initiation. Ca2+, as an important secondary messenger, plays a central role in polarized cell growth including cotton fiber elongation. However, the role of Ca2+ in fiber initiation is far from well understood. In this paper, through ovule culture we demonstrate that Ca2+ is crucial for fiber initiation. Using transgenic cotton expressing the fluorescent Ca2+ indicator YC3.60, we show cellular and intracellular distribution of Ca2+ in cotton ovule epidermis and fiber cells. In the initiating fiber cell, Ca2+ accumulated mainly at the base of the cell, while in the fast elongating cell, the Ca2+ was enriched in the tip region. This cellular distribution of Ca2+ reported by YC3.60 was confirmed by the staining with a Ca2+-sensitive dye fluo-3/AM. Compared to the fluorescent dye staining, the YC3.60 system can reveal more detailed information on the intracellular distribution without photobleaching. Taken together, our data suggest that Ca2+ plays an important role in spherical expansion of cotton fiber initials.