Elevation Of Intracellular Calcium Concentration Research Articles

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

An insight into the mechanistic role of (-)-Ampelopsin F from Vatica chinensis L. in inducing insulin secretion in pancreatic beta cells

Resveratrol oligomers, ranging from dimers to octamers, are formed through regioselective synthesis involving the phenoxy radical coupling of resveratrol building blocks, exhibiting remarkable therapeutic potential, including antidiabetic properties. In this study, we elucidate the mechanistic insights into the insulin secretion potential of a resveratrol dimer, (-)-Ampelopsin F (AmF), isolated from the acetone extract of Vatica chinensis L. stem bark in Pancreatic Beta-TC-6 cell lines. The AmF (50 µM) treated cells exhibited a 3.5-fold increase in insulin secretion potential as compared to unstimulated cells, which was achieved through the enhancement of mitochondrial membrane hyperpolarization, elevation of intracellular calcium concentration, and upregulation of GLUT2 and glucokinase expression in pancreatic Beta-TC-6 cell lines. Furthermore, AmF effectively inhibited the activity of DPP4, showcasing a 2.5-fold decrease compared to the control and a significant 6.5-fold reduction compared to the positive control. These findings emphasize AmF as a potential lead for the management of diabetes mellitus and point to its possible application in the next therapeutic initiatives.

Recent advances in the structure and activation mechanisms of metabolite-releasing Pannexin 1 channels.

Pannexin 1 (PANX1) is a widely expressed large-pore ion channel located in the plasma membrane of almost all vertebrate cells. It possesses a unique ability to act as a conduit for both inorganic ions (e.g. potassium or chloride) and bioactive metabolites (e.g. ATP or glutamate), thereby activating varying signaling pathways in an autocrine or paracrine manner. Given its crucial role in cell-cell interactions, the activity of PANX1 has been implicated in maintaining homeostasis of cardiovascular, immune, and nervous systems. Dysregulation of PANX1 has also been linked to numerous diseases, such as ischemic stroke, seizure, and inflammatory disorders. Therefore, the mechanisms underlying different modes of PANX1 activation and its context-specific channel properties have gathered significant attention. In this review, we summarize the roles of PANX1 in various physiological processes and diseases, and analyze the accumulated lines of evidence supporting diverse molecular mechanisms associated with different PANX1 activation modalities. We focus on examining recent discoveries regarding PANX1 regulations by reversible post-translational modifications, elevated intracellular calcium concentration, and protein-protein interactions, as well as by irreversible cleavage of its C-terminal tail. Additionally, we delve into the caveats in the proposed PANX1 gating mechanisms and channel open-closed configurations by critically analyzing the structural insights derived from cryo-EM studies and the unitary properties of PANX1 channels. By doing so, we aim to identify potential research directions for a better understanding of the functions and regulations of PANX1 channels.

Leveraging Plasma Membrane Repair Therapeutics for Treating Neurodegenerative Diseases.

Plasma membrane repair is an essential cellular mechanism that reseals membrane disruptions after a variety of insults, and compromised repair capacity can contribute to the progression of many diseases. Neurodegenerative diseases are marked by membrane damage from many sources, reduced membrane integrity, elevated intracellular calcium concentrations, enhanced reactive oxygen species production, mitochondrial dysfunction, and widespread neuronal death. While the toxic intracellular effects of these changes in cellular physiology have been defined, the specific mechanism of neuronal death in certain neurodegenerative diseases remains unclear. An abundance of recent evidence indicates that neuronal membrane damage and pore formation in the membrane are key contributors to neurodegenerative disease pathogenesis. In this review, we have outlined evidence supporting the hypothesis that membrane damage is a contributor to neurodegenerative diseases and that therapeutically enhancing membrane repair can potentially combat neuronal death.

Calpain-mediated Mechanoptosis in Breast Adenocarcinoma.

Calpains belong to a family of important calcium-dependent cysteine proteases. They are involved in intracellular processes including cytoskeleton disorganization and substrate proteolysis. They also enhance apoptosis and cell to cell adhesion. Calpains demonstrate also a mechanosensory function in neoplastic and malignant cells due to their implication in mechanoptosis. This is a specific type of apoptotic death induced by strong external mechanical stimuli. Anti-cytoskeleton rigidity inhibition strategies based on calpain induction lead to increased apoptosis of tumor transformed cells. Elevated intracellular calcium concentration mediated by specific receptors and channels activates calpains. In the current molecular review, we explored the role of calpains in calcium-dependent signa transduction pathways in breast adenocarcinoma in conjunction with novel agents that activate their important anti-tumor functions.

MicroRNA-423-5p Mediates Cocaine-Induced Smooth Muscle Cell Contraction by Targeting Cacna2d2.

Cocaine abuse increases the risk of atherosclerotic cardiovascular disease (CVD) and causes acute coronary syndromes (ACS) and hypertension (HTN). Significant research has explored the role of the sympathetic nervous system mediating the cocaine effects on the cardiovascular (CV) system. However, the response of the sympathetic nervous system alone is insufficient to completely account for the CV consequences seen in cocaine users. In this study, we examined the role of microRNAs (miRNAs) in mediating the effect of cocaine on the CV system. MiRNAs regulate many important biological processes and have been associated with both response to cocaine and CV disease development. Multiple miRNAs have altered expression in the CV system (CVS) upon cocaine exposure. To understand the molecular mechanisms underlying the cocaine response in the CV system, we studied the role of miRNA-423-5p and its target Cacna2d2 in the regulation of intracellular calcium concentration and SMC contractility, a critical factor in the modulation of blood pressure (BP). We used in vivo models to evaluate BP and aortic stiffness. In vitro, cocaine treatment decreased miR-423-5p expression and increased Cacna2d2 expression, which led to elevated intracellular calcium concentrations and increased SMC contractility. Overexpression of miR-423-5p, silencing of its target Cacna2d2, and treatment with a calcium channel blocker reversed the elevated SMC contractility caused by cocaine. In contrast, suppression of miR-423-5p increased the intracellular calcium concentration and SMC contractibility. In vivo, smooth muscle-specific overexpression of miR-423-5p ameliorated the increase in BP and aortic stiffness associated with cocaine use. Thus, miR-423-5p regulates SMC contraction by modulating Cacna2d2 expression increasing intracellular calcium concentrations. Modulation of the miR-423-5p-Cacna2d2-Calcium transport pathway may represent a novel therapeutic strategy to improve cocaine-induced HTN and aortic stiffness.

Clioquinol induces autophagy by down-regulation of calreticulin in human neurotypic SH-SY5Y cells

Clioquinol (CQ) is considered as a promising drug of neurodegenerative diseases. However, the underlying mechanism is unclear. Our previous study has proved that CQ induces S-phase cell cycle arrest through the elevation of intracellular calcium concentration ([Ca2+]i) with high levels of SERCA2. Furthermore, it could induce autophagy in an intracellular calcium independent manner in human neurotypic SH-SY5Y cells. In this study, the involvement of calreticulin (CRT) in autophagy induced by CQ was investigated. Our results illustrated the endoplasmic reticulum (ER) stress induced by CQ and DTT led to the cell death in different manners. DTT, an ER stress positive control, induced UPR accompanied with up-regulation of CRT and apoptosis, while CQ inhibited UPR accompanied with down-regulation of CRT，resulting in autophagy. Then, overexpression of CRT was shown to cause UPR and decrease [Ca2+]i, leading to cell apoptosis and inhibition of S-phase arrest induced by CQ. While the UPR was alleviated and autophagy was further enhanced in CRT deficient cells by using targeted siRNA. Meanwhile, down-regulation of CRT resulted in [Ca2+]i overload and induction of S-phase arrest. Finally, we found that the effect of CQ on the HT22 cells was similar to that on the SH-SY5Y cells. Our data showed for the first time that CQ decreased expression of CRT, leading to autophagy, an increase of [Ca2+]i, and cell S-phase arrest in the neurotypic cells. The present study describes the cellular signal pathways regulating autophagy by CQ and highlights the potential therapeutic application of CQ in neurodegenerative disorders.

Can KCa3.1 channel activators serve as novel inhibitors of platelet aggregation?

The intermediate‐conductance, calcium‐activated potassium channel KCa3.1, first cloned in 1997,1 is also known as IK, IKCa, SK4, KCNN4 or due to its role in erythrocyte volume regulation as the Gárdos channel.2,3 KCa3.1 channels are widely expressed in non‐excitable tissues such as red and white blood cells, proliferating smooth muscle and fibroblasts, vascular endothelium, and secretory epithelia.4 Based on their ability to hyperpolarize the cell membrane upon elevation of intracellular calcium concentration ([Ca2+]i), KCa3.1 channels regulate calcium signaling and downstream processes such as activation, proliferation, and secretion.

A revised calcium-dependent model of transcranial magnetic theta-burst stimulation

ObjectiveCalcium dependency is presently an essential assumption in modelling the neuromodulatory effects of transcranial magnetic stimulation. Y.Z.Huang et al.developed the first neuromodulation model to explain the bidirectional effects of theta-burst stimulation (TBS) based on the postsynaptic intracellular calcium concentration elevation. However, we discover that the published computer code is not consistent with the model formulation, neither do the parameters and derived plots consequently match the formulations. Here we intend to fix the computer code and re-calibrate the model. MethodsWe corrected the affected difference equations and re-calibrated the revised model with experimental data using non-convex optimisation based on a L2 penalty. ResultsThe revised model outperforms the initial model in characterising the relative motor-evoked potential levels of TBS-induced after-effects in various conditions. ConclusionsWe corrected the inconsistencies in the previous model and computer code and provided a complete calibration to support the research that is based on it. SignificanceThis work improves the accuracy and secures the scope of the model, which is necessary to retain a rich body of research resulting from the model. Furthermore, this model provides both a quantitative model for several parameters of TBS and a basic model foundation for future refinement.

Elevated and Sustained Intracellular Calcium Signalling Is Necessary for Efficacious Induction of the Human Sperm Acrosome Reaction.

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.

The KCNQ channel inhibitor XE991 suppresses nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglion neurons.

XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone) is reportedly a potent and selective Kv7 (KCNQ) channel inhibitor. This study aimed to evaluate how XE991 affects nicotinic responses in intracardiac ganglion neurons. We studied how the KCNQ channel inhibitor XE991 could affect nicotinic responses in acutely isolated rat intracardiac ganglion neurons using a perforated patch-clamp recording configuration and Ca2+ imaging. XE991 reversibly and concentration-dependently inhibited the nicotine (10μM)-induced current with an IC50 of 14.4μM. The EC50 values for nicotine-induced currents in the absence and presence of 10μM XE991 were 8.7 and 12.0μM, respectively. Because XE991 suppressed the maximum response of the nicotine concentration-response curve, the inhibitory effect of this drug appears to be noncompetitive. In addition, linopirdine reduced the amplitude of 10µM nicotine-induced currents with an IC50 value of 16.9μM. The inorganic KCNQ channel inhibitor Ba2+ affected neither the nicotine-induced current nor the inhibitory effect of XE991 on the nicotinic response. The KCNQ activator flupirtine at a concentration of 10μM slightly but markedly inhibited the nicotine-induced current. Finally, XE991 inhibited the nicotine-induced elevation of intracellular calcium concentration and the nicotine-induced firing of action potentials. We propose that XE991 inhibits nicotinic acetylcholine receptors in intracardiac ganglion neurons, which in turn attenuate nicotine-induced neuronal excitation.

Can Virus-like Particles Be Used as Synergistic Agent in Pest Management?

Among novel strategies proposed in pest management, synergistic agents are used to improve insecticide efficacy through an elevation of intracellular calcium concentration that activates the calcium-dependent intracellular pathway. This leads to a changed target site conformation and to increased sensitivity to insecticides while reducing their concentrations. Because virus-like particles (VLPs) increase the intracellular calcium concentration, they can be used as a synergistic agent to synergize the effect of insecticides. VLPs are self-assembled viral protein complexes, and by contrast to entomopathogen viruses, they are devoid of genetic material, which makes them non-infectious and safer than viruses. Although VLPs are well-known to be used in human health, we propose in this study the development of a promising strategy based on the use of VLPs as synergistic agents in pest management. This will lead to increased insecticides efficacy while reducing their concentrations.

Sodium butyrate potentiates insulin secretion from rat islets at the expense of compromised expression of \u03b2 cell identity genes

Short-chain fatty acids (SCFAs) produced by the gut microbiota have been well demonstrated to improve metabolic homeostasis. However, the role of SCFAs in islet function remains controversial. In the present study, none of the sodium acetate, sodium propionate, and sodium butyrate (SB) displayed acute impacts on insulin secretion from rat islets, whereas long-term incubation of the three SCFAs significantly potentiated pancreatic β cell function. RNA sequencing (RNA-seq) revealed an unusual transcriptome change in SB-treated rat islets, with the downregulation of insulin secretion pathway and β cell identity genes, including Pdx1, MafA, NeuroD1, Gck, and Slc2a2. But these β cell identity genes were not governed by the pan-HDAC inhibitor trichostatin A. Overlapping analysis of H3K27Ac ChIP-seq and RNA-seq showed that the inhibitory effect of SB on the expression of multiple β cell identity genes was independent of H3K27Ac. SB treatment increased basal oxygen consumption rate (OCR), but attenuated glucose-stimulated OCR in rat islets, without altering the expressions of genes involved in glycolysis and tricarboxylic acid cycle. SB reduced the expression of Kcnj11 (encoding KATP channel) and elevated basal intracellular calcium concentration. On the other hand, SB elicited insulin gene expression in rat islets through increasing H3K18bu occupation in its promoter, without stimulating CREB phosphorylation. These findings indicate that SB potentiates islet function as a lipid molecule at the expense of compromised expression of islet β cell identity genes.

A Negative Feedback Loop in Ultraviolet A-Induced Senescence in Human Dermal Fibroblasts Formed by SPCA1 and MAPK.

Secretory pathway calcium ATPase 1 (SPCA1) is a calcium pump localized specifically to the Golgi. Its effects on UVA-induced senescence have never been examined. In our study, expression of SPCA1 was increased in UVA-irradiated human dermal fibroblasts (HDFs) by activating mitogen-activated protein kinase (MAPK) and its downstream transcription factor, c-jun. Dual-luciferase reporter and chromatin immunoprecipitation assays revealed that c-jun regulated SPCA1 by binding to its promoter. Furthermore, downregulating SPCA1 with siRNA transfection aggravated UVA-induced senescence due to an elevation of intracellular calcium concentrations and a subsequent increase in reactive oxygen species (ROS) and MAPK activity. In contrast, overexpression of SPCA1 reduced calcium overload, consequently lowering the ROS level and suppressing MAPK activation. This alleviated the cellular senescence caused by UVA irradiation. These results indicated that SPCA1 might exert a protective effect on UVA-induced senescence in HDFs via forming a negative feedback loop. Specifically, activation of MAPK/c-jun triggered by UVA transcriptionally upregulated SPCA1. In turn, the increased SPCA1 lowered the intracellular Ca2+ level, probably through pumping Ca2+ into the Golgi, leading to a reduction of ROS, eventually decreasing MAPK activity and diminishing UVA-induced senescence.

The Ryanodine Receptor Contributes to the Lysophosphatidylcholine-Induced Mineralization in Valvular Interstitial Cells.

Fibrocalcific aortic valve disease (CAVD) is caused by the deposition of calcific nodules in the aortic valve leaflets, resulting in progressive loss of function that ultimately requires surgical intervention. This process is actively mediated by the resident valvular interstitial cells (VICs), which, in response to oxidized lipids, transition from a quiescent to an osteoblast-like state. The purpose of this study was to examine if the ryanodine receptor, an intracellular calcium channel, could be therapeutically targeted to prevent this phenotypic conversion. The expression of the ryanodine receptor in porcine aortic VICs was characterized by qRT-PCR and immunofluorescence. Next, the VICs were exposed to lysophosphatidylcholine, an oxidized lipid commonly found in low-density lipoprotein, while the activity of the ryanodine receptor was modulated with ryanodine. The cultures were analyzed for markers of cellular mineralization, alkaline phosphatase activity, proliferation, and apoptosis. Porcine aortic VICs predominantly express isoform 3 of the ryanodine receptors, and this protein mediates the cellular response to LPC. Exposure to LPC caused elevated intracellular calcium concentration in VICs, raised levels ofalkaline phosphatase activity, and increased calcific nodule formation, but these changes were reversed when the activity of the ryanodine receptor was blocked. Our findings suggest blocking the activity of the ryanodine receptor can attenuate the valvular mineralization caused by LPC. We conclude that oxidized lipids, such as LPC, play an important role in the development and progression of CAVD and that the ryanodine receptor is a promising target for pharmacological intervention.

Perfluorooctane sulfonate acute exposure stimulates insulin secretion via GPR40 pathway

Perfluoroalkyl substances (PFASs) are widely used synthetic chemicals, showing environmental/biological persistence and adverse effects on ecosystem and human health. Several epidemiological and animal studies have revealed that PFASs levels are associated with elevated serum insulin level; however, the effect of PFASs on insulin secretion and the underlying mechanism are not clear. In this study, the effect of a most concerned PFAS, perfluorooctane sulfonate (PFOS) on insulin secretion in Beta-TC-6 pancreatic cells was studied. The results showed that PFOS acute exposure stimulated insulin secretion and elevated intracellular calcium concentration ([Ca2+]i). The PFOS-stimulated [Ca2+]i elevation was resulted from both extra- and intra-cellular sources. PFOS acute exposure decreased ATP content and ATP/ADP ratio, indicating the mitochondrial function was damaged under PFOS acute exposure. The PFOS-stimulated insulin secretion was inhibited by GW1100, a G Protein-coupled Receptor 40 (GPR40) specific inhibitor, but not affected by GW9662, a specific antagonist to the peroxisome proliferator-activated receptor gamma (PPARγ). The observation of RNA silencing further demonstrated that the PFOS-stimulated insulin secretion is, at least partially, via GPR40. By using specific inhibitors, we found that the GPR40 downstream pathways, phospholipase C (PLC) and L-type Ca2+ channels (LTCC) were involved in PFOS-stimulated [Ca2+]i elevation and insulin secretion.

Abstract B63: Calcium electroporation offers an effective local treatment with immunogenic cell death and the potential for systemic remission in melanoma

Abstract Electrochemotherapy is an established treatment modality in which chemotherapeutics are delivered systemically or locally in combination with local electrical pulses. Pulsing parameters are optimized to induce the reversible formation of pores in the cell membrane, leaving cells intact and healthy. Osmotic gradients during poration result in the rapid uptake of extracellular components, which results in enhanced chemotherapeutic uptake, between 5 to 2,000-fold depending on the hydrophobicity of the drug used. Complete local response rates for dermal lesions exceed 80%. Treatment is suitable for use in proximity to vasculature and offers superior cosmetic results in comparison to surgery when used in areas such as the eyelid or mouth. Recent advances have, however, been able to replace cytotoxic chemotherapeutic agents with calcium with no decrease in efficacy. The nature of cell death induced is believed to be acute, but to date has not been clearly characterized. Using Flou-4 to track the duration of elevated intracellular calcium concentrations, we have shown that elevated calcium levels persist for hours following treatment. Cell death occurs in 60-80% of cells treated in vitro within 8 hours of treatment. Cells surviving this initial insult show no detectable loss in survival or growth potential and appear otherwise unaffected. Necrotic cell death provides a significant source of damage-associated molecular patterns and may aid the generation of anticancer immune responses. Using cocultured macrophages, we have been able to show that treatment can indirectly boost cytotoxic T-cell responses via tumor-associated macrophages. The elucidation of cell death during treatment is critical for the effective optimization of clinical treatment parameters, which has hitherto been determined empirically. Other advances, such as the use of high-frequency electroporation, are transforming the clinical delivery. It is foreseeable that the requirement of surgical theaters may be avoided with the development of a pain-free treatment in the absence of cytotoxic drugs. Note:This abstract was not presented at the conference. Citation Format: Liam Friel Tremble, Cynthia Heffron, Patrick Forde. Calcium electroporation offers an effective local treatment with immunogenic cell death and the potential for systemic remission in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B63.

Ability of plasma-activated acetated Ringer's solution to induce A549 cell injury is enhanced by a pre-treatment with histone deacetylase inhibitors.

Non-thermal plasma (NTP) is applicable to living cells and has emerged as a novel technology for cancer therapy. NTP affect cells not only by direct irradiation, but also by an indirect treatment with previously prepared plasma-activated liquid. Histone deacetylase (HDAC) inhibitors have the potential to enhance susceptibility to anticancer drugs and radiation because these reagents decondense the compact chromatin structure by neutralizing the positive charge of the histone tail. The aim of the present study was to demonstrate the advantage of the combined application of plasma-activated acetated Ringer’s solution (PAA) and HDAC inhibitors on A549 cancer cells. PAA maintained its ability for at least 1 week stored at any temperature tested. Cell death was enhanced more by combined regimens of PAA and HDAC inhibitors, such as trichostatin A (TSA) and valproic acid (VPA), than by a single PAA treatment and was accompanied by ROS production, DNA breaks, and mitochondria dysfunction through a caspase-independent pathway. These phenomena induced the depletion of ATP and elevations in intracellular calcium concentrations. The sensitivities of HaCaT cells as normal cells to PAA were less than that of A549 cells. These results suggest that HDAC inhibitors synergistically induce the sensitivity of cancer cells to PAA.

P299 Acute effects of Levosimendan on myocardial function in patients with severe mitral regurgitation and left ventricular dysfunction undergoing MitraClip repair

Abstract Background MitraClip system has developed as a valid therapeutic option in patients affected by moderate to severe and severe mitral regurgitation, low left ventricular ejection fraction (LVEF) and high surgical risk. Often, after the procedure occurs afterload mismatch, an acute and transient worsening of LVEF. Inotropic drugs can improve hemodynamic values at the prize of severe side effects. Levosimendan increases myocardial contractility without an elevation of intracellular calcium concentration, tachyarrhythmia and cardiomyocytes necrosis. Purpose Aim of our study was to assess the acute Levosimendan effects on LVEF of patients who underwent MitraClip procedure Methods Among 160 patients who underwent MitraClip procedure in our institute, 99 patients, with LVEF ≤35%, were included in the study. Transthoracic echocardiogram was performed in all patients, at moment of hospital admission and at discharge; transesophageal echocardiogram was performed during the procedure. We recorded the LVEF by modified Simpson’s rule. Periprocedural hemodynamic parameters were also recorded. 59 patients received Levosimendan during and early after the procedure (L-group) and 40 patients did not (no-L-group). Levosimendan perfusion was started at 0.01 μg/kg/min 1 h before the procedure without a loading dose, and maintained for 12h, according to hemodynamics. Results In the overall population, patients suffered from a severe reduction of LVEF (29.5 ± 5.3%) and high systolic pulmonary arterial pressure (sPAP) (51 ± 14.2 mmHg), without significant difference between the two groups. Acute procedural success was achieved in 98% of the study population, with 2 procedural failures in no-L-group (p = 0.16). During the procedure we observed a significant improvement of LVEF compared to baseline values only in L-group (from 29.6 ± 5.7% to 32.1 ± 7.6%, p = 0.046); in no-L-group the LVEF improved from 29.4 ± 5% to 30.2 ± 4.9% (p = 0.47); at discharge the LVEF was 31.3 ± 4.9% and 30.8 ± 5.7%, in L-group and no-L-group, respectively (p = ns compared to baseline and procedure). At discharge the sPAP significantly reduced in the overall population to 46.3 ± 12.7 mmHg (p 0.015): from 50.8 ± 12.3 mmHg vs 48.7 ± 11.9 in L-group (p = 0.35); from 51.2 ± 16 to 44.3 ± 13.2 mmHg (p = 0.04) in no-L-group. In-hospital mortality was 1.7% in L-group (1 patient die) and 0% in no-L-group. No relevant arrhythmias were reported in any patient during the hospital recovery. Conclusion In MitraClip patients with severe reduction of LVEF, Levosimendan has proven to improve hemodynamic outcome, increasing myocardial contractility during and early after procedure.

Using fluorescent calcium indicators in neuronal ion channel studies

Calcium ion (Ca2+) is the most prominent secondary messenger of physiological cellular signals and ion channels research (Moeder et al. 2019). Neurotransmission, muscle contraction and fertilization are only a few of the physiological properties that make calcium ion important in most eukaryotic cells (Pharris et al.  2018). The extracellular calcium concentration is 1-3 mM whereas the intracellular concentration is around 80-100 nM. This tremendous difference is tightly controlled by dozens of different ion channels embedded in the membrane (Van Hook et al.  2019). Activation of these channels causes calcium ions to entry into the cell with density gradient. This produce the calcium signal. It is very important to control that prolonged elevation of intracellular calcium concentration due to channelopathies in ion channels due to various nervous system diseases because it activates caspase cascades leading to permanent damage and apoptosis. In this respect, the calcium indicators are unrivaled in terms of taking clear results by photographing all the points in these steps from the stage they were developed to the final stages. The use of these indicators to address the roles of these ion channels in terms of their contribution to the pathogenesis of neurological diseases has been an indispensible method of molecular analysis in recent years (Xu and Dong, 2019). In this presentation, we will discuss the chemical structures of calcium indicators, their contribution to the examination of cellular signaling pathways, their advantages and disadvantages in the investigation of ion channels in neurological diseases.