Extracellular Calcium Concentration Research Articles

Functional evaluation of a novel nonsense variant of the calcium-sensing receptor gene leading to hypocalcemia.

The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.

Hematin- and Hemin-Induced Spherization and Hemolysis of Human Erythrocytes Are Independent of Extracellular Calcium Concentration.

Pathologies such as malaria, hemorrhagic stroke, sickle cell disease, and thalassemia are characterized by the release of hemoglobin degradation products from damaged RBCs. Hematin (liganded with OH-) and hemin (liganded with Cl-)-are the oxidized forms of heme with toxic properties due to their hydrophobicity and the presence of redox-active Fe3. In the present study, using the original LaSca-TM laser particle analyzer, flow cytometry, and confocal microscopy, we showed that both hematin and hemin induce dose-dependent RBC spherization and hemolysis with ghost formation. Hematin and hemin at nanomolar concentrations increased [Ca2+]i in RBC; however, spherization and hemolysis occurred in the presence and absence of calcium, indicating that both processes are independent of [Ca2+]i. Both compounds triggered acute phosphatidylserine exposure on the membrane surface, reversible after 60 min of incubation. A comparison of hematin and hemin effects on RBCs revealed that hematin is a more reactive toxic metabolite than hemin towards human RBCs. The toxic effects of heme derivatives were reduced and even reversed in the presence of albumin, indicating the presence in RBCs of the own recovery system against the toxic effects of heme derivatives.

Contractions Induced in Human Pulmonary Arteries by a H2S Donor, GYY 4137, Are Inhibited by Low-Frequency (20 kHz) Ultrasound.

The present study aimed to investigate the effect of a H2S donor, GYY 4137, on human pulmonary arteries and whether low-frequency ultrasound (20 kHz, 4 W/cm2) inhibits GYY 4137 contractions. Functional studies were conducted on human and rat pulmonary arteries mounted on microvascular myographs. We placed an ultrasonic gadget in the tissue organ bath to insonate the arteries with low-frequency ultrasound. To measure the effect of the low-frequency ultrasound on the entrance of extracellular Ca2+, the preparations were placed in a Ca2+-free solution, and the thromboxane agonist, U46619, and extracellular calcium were added in the presence of insonation. In isolated human pulmonary arteries, GYY 4137 induced contractions, which were most pronounced in the arteries contracted with the thromboxane analogue, U46619. The transient GYY4137 contractions were reversed by low-frequency ultrasound, a blocker of KV7 channels, XE-991 (10 µM), and glibenclamide (1 μM), a blocker of ATP-sensitive channels. Low-frequency ultrasound also inhibited the contractions induced by the smooth muscle entrance of increasing extracellular calcium concentrations. The present findings show that GYY 4137 can cause a transient contraction of pulmonary arteries in human arteries. GYY 4137 alone does not cause significant vascular contraction in rat lung arteries, but it contracts rat lung arteries precontracted with U46619. The transient contractions induced by GYY 4137 can be inhibited by low-frequency ultrasound, probably by counteracting the influx of external Ca2+. The effect of low-frequency ultrasound counteracts contraction in pulmonary arteries; therefore, a possibility could be to develop a larger device allowing treatment of patients with pulmonary hypertension.

Effects of Extracellular Calcium Concentration on Hepatic Ischemia-Reperfusion Injury in a Rat Model.

Hypocalcemia is frequently identified during liver transplant. However, supplementation of extracellular calcium could induce increased intracellular calcium concentration, as a potential factor for injury to the liver graft. We evaluated the effects of regulating extracellular calcium concentrations on hepatic ischemia-reperfusion injury. We randomly divided 24 Sprague-Dawley rats into 3 groups: group C received normal saline (n = 8), group L received citrate to induce hypocalcemia (n = 8), and group L-Co received citrate followed by calcium gluconate to ameliorate hypocalcemia (n = 8). Liver enzyme levels and extracellular calcium were measured before surgery, 1 hour after ischemia, and 2 hours after reperfusion. The primary outcome was liver enzyme levels measured 2 hours after reperfusion. In addition, we evaluated intracellular calcium levels, lactate dehydrogenase activity, and histopathological results in liver tissue. Three groups demonstrated significant differences in extracellular calcium concentrations, but intracellular calcium concentrations in liver tissue were not significantly different. Group L showed significantly lower mean arterial pressure than other groups at 1 hour after ischemia (93.6 ± 20.8 vs 69.4 ± 14.2 vs 86.6 ± 10.4 mmHg; P = .02, for group C vs L vs L-Co, respectively). At 2 hours after reperfusion, group L showed significantly higher liver enzymes than other groups (aspartate aminotransferase 443.0 ± 353.2 vs 952.3 ± 94.8 vs 502.4 ± 327.3 U/L, P = .01; and alanine aminotransferase 407.9 ± 406.5 vs 860.6 ± 210.9 vs 333.9 ± 304.2 U/L, P = .02; for group C vs L vs L-Co, respectively). However, no significant difference was shown in lactate dehydrogenase and histological liver injury grade. Administering calcium to rats with hypocalcemia did not increase intracellular calcium accumulation but instead resulted in less hepatic injury compared with rats with low extracellular calcium concentrations in this rat model study.

Low-molecular-weight fucoidan ameliorates calcium-deficient-diet–induced bone loss and osteoarthritis via CaSR level regulation

Fucoidans—sulfated and fucosylated polysaccharides extracted from brown seaweed—reportedly accelerate bone growth and prevent osteoarthritis (OA) progression. In this study, we examined the effects of low-molecular-weight fucoidan (LMF) on the molecular mechanism of extracellular calcium concentration in vitro and in vivo. In MG63 cells, LMF significantly increased the activity of alkaline phosphatase (ALP) and expression of calcium-sensing receptor (CaSR), runt-related transcription factor 2 (Runx2), and osteoprotegerin (OPG) under low- and normal-[Ca+2]0 conditions, but only decreased the expression of receptor activator of the nuclear factor κB ligand (RANKL) under normal-[Ca+2]0 conditions. In the calcium-deficient diet model, LMF significantly increased body length, growth rate, bone-specific ALP secretion, trabecular number, and CaSR protein expression, without body weight changes. In the OA model, LMF significantly increased bone volume and CaSR protein expression and reduced joint inflammation. Overall, our results indicate that LMF reduced calcium-deficient-diet–induced bone loss and OA via CaSR level regulation.

Neurovirulent cytokines increase neuronal excitability in a model of coronavirus-induced neuroinflammation

BackgroundNeurological manifestations of severe coronavirus infections, including SARS-CoV-2, are wide-ranging and may persist following virus clearance. Detailed understanding of the underlying changes in brain function may facilitate the identification of therapeutic targets. We directly tested how neocortical function is impacted by the specific panel of cytokines that occur in coronavirus brain infection. Using the whole-cell patch-clamp technique, we determined how the five cytokines (TNFα, IL-1β, IL-6, IL-12p40 and IL-15 for 22–28-h) at concentrations matched to those elicited by MHV-A59 coronavirus brain infection, affected neuronal function in cultured primary mouse neocortical neurons.ResultsWe evaluated how acute cytokine exposure affected neuronal excitability (propensity to fire action potentials), membrane properties, and action potential characteristics, as well as sensitivity to changes in extracellular calcium and magnesium (divalent) concentration. Neurovirulent cytokines increased spontaneous excitability and response to low divalent concentration by depolarizing the resting membrane potential and hyperpolarizing the action potential threshold. Evoked excitability was also enhanced by neurovirulent cytokines at physiological divalent concentrations. At low divalent concentrations, the change in evoked excitability was attenuated. One hour after cytokine removal, spontaneous excitability and hyperpolarization of the action potential threshold normalized but membrane depolarization and attenuated divalent-dependent excitability persisted.ConclusionsCoronavirus-associated cytokine exposure increases spontaneous excitability in neocortical neurons, and some of the changes persist after cytokine removal.

Nitric oxide modulates NMDA receptor through a negative feedback mechanism and regulates the dynamical behavior of neuronal postsynaptic components

Nitric oxide (NO) is known to be an important regulator of neurological processes in the central nervous system which acts directly on the presynaptic neuron and enhances the release of neurotransmitters like glutamate into the synaptic cleft. Calcium influx activates a cascade of biochemical reactions to influence the production of nitric oxide in the postsynaptic neuron. This has been modeled in the present work as a system of ordinary differential equations, to explore the dynamics of the interacting components and predict the dynamical behavior of the postsynaptic neuron. It has been hypothesized that nitric oxide modulates the NMDA receptor via a feedback mechanism and regulates the dynamic behavior of postsynaptic components. Results obtained by numerical analyses indicate that the biochemical system is stimulus-dependent and shows oscillations of calcium and other components within a limited range of concentration. Some of the parameters such as stimulus strength, extracellular calcium concentration, and rate of nitric oxide feedback are crucial for the dynamics of the components in the postsynaptic neuron.

High calcium concentrations reduce cellular excitability of mouse MNTB neurons

Calcium, a universal intracellular signaling molecule, plays essential roles in neural functions. Historically, in most in vitro brain slice electrophysiology studies, the extracellular calcium concentration ([Ca2+]e) in artificial cerebrospinal fluid is of a wide range and typically higher than the physiological value. At high [Ca2+]e, synaptic transmission is generally enhanced. However, the effects and the underlying mechanisms of calcium on intrinsic neuronal properties are diverse. Using whole-cell patch clamp in acute brainstem slices obtained from mice of either sex, we investigated the effects and the underlying mechanisms of high [Ca2+]e on intrinsic neuronal properties of neurons in the medial nucleus of the trapezoid body (MNTB), an auditory brainstem component in the sound localization circuitry. Compared to the physiological [Ca2+]e (1.2 mM), high [Ca2+]e at 1.8 and 2.4 mM significantly reduced the cellular excitability of MNTB neurons, resulting in decreased spike firing rate, depolarized spike threshold, and decreased the ability to follow high frequency inputs. High extracellular magnesium concentrations at 1.8 and 2.4 mM produced similar but less robust effects, due to surface charge screening. Upon high calcium application, voltage-gated sodium channel currents remained largely unchanged. Calcium-sensing receptors were detected in MNTB neurons, but blocking these receptors did not eliminate the effects of high calcium on spontaneous spiking. We attribute the lack of significant effects in these last two experiments to the moderate changes in calcium we tested. Our results call for the use of physiological [Ca2+]e in brain slice experiments.

Contributions of the Sodium Leak Channel NALCN to Pacemaking of Medial Ventral Tegmental Area and Substantia Nigra Dopaminergic Neurons.

We tested the role of the sodium leak channel, NALCN, in pacemaking of dopaminergic neuron (DAN) subpopulations from adult male and female mice. In situ hybridization revealed NALCN RNA in all DANs, with lower abundance in medial ventral tegmental area (VTA) relative to substantia nigra pars compacta (SNc). Despite lower relative abundance of NALCN, we found that acute pharmacological blockade of NALCN in medial VTA DANs slowed pacemaking by 49.08%. We also examined the electrophysiological properties of projection-defined VTA DAN subpopulations identified by retrograde labeling. Inhibition of NALCN reduced pacemaking in DANs projecting to medial nucleus accumbens (NAc) and others projecting to lateral NAc by 70.74% and 31.98%, respectively, suggesting that NALCN is a primary driver of pacemaking in VTA DANs. In SNc DANs, potentiating NALCN by lowering extracellular calcium concentration speeded pacemaking in wildtype but not NALCN conditional knockout mice, demonstrating functional presence of NALCN. In contrast to VTA DANs, however, pacemaking in SNc DANs was unaffected by inhibition of NALCN. Instead, we found that inhibition of NALCN increased the gain of frequency-current plots at firing frequencies slower than spontaneous firing. Similarly, inhibition of the hyperpolarization-activated cyclic nucleotide-gated (HCN) conductance increased gain but had little effect on pacemaking. Interestingly, simultaneous inhibition of NALCN and HCN resulted in significant reduction in pacemaker rate. Thus, we found NALCN makes substantial contributions to driving pacemaking in VTA DAN subpopulations. In SNc DANs, NALCN is not critical for pacemaking but inhibition of NALCN makes cells more sensitive to hyperpolarizing stimuli.SIGNIFICANCE STATEMENT Pacemaking in midbrain dopaminergic neurons (DAN) relies on multiple subthreshold conductances, including a sodium leak. Whether the sodium leak channel, NALCN, contributes to pacemaking in DANs located in the VTA and the SNc has not yet been determined. Using electrophysiology and pharmacology, we show that NALCN plays a prominent role in driving pacemaking in projection-defined VTA DAN subpopulations. By contrast, pacemaking in SNc neurons does not rely on NALCN. Instead, the presence of NALCN regulates the excitability of SNc DANs by reducing the gain of the neuron's response to inhibitory stimuli. Together, these findings will inform future efforts to obtain DAN subpopulation-specific treatments for use in neuropsychiatric disorders.

Pharmacological profile of upacicalcet, a novel positive allosteric modulator of calcium-sensing receptor, in vitro and in vivo

Upacicalcet (formerly SK-1403/AJT240) is a novel non-peptide calcimimetic agent that acts as a calcium-sensing receptor (CaSR) agonist for the treatment of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). We compared upacicalcet with other calcimimetics (etelcalcetide or cinacalcet) and examined its in vitro and in vivo characteristics in terms of its human CaSR agonistic activity, its efficacy in normal and CKD rats after a single administration, and its effect on gastric emptying in rats. Upacicalcet activated human CaSR depending on the extracellular calcium (Ca2+) concentration without exhibiting an agonistic action when the extracellular Ca2+ level was below the physiological level. On the other hand, etelcalcetide had an agonistic activity even in the absence of physiological levels of extracellular Ca2+. The intravenous administration of upacicalcet to normal and double-nephrectomized rats dose-dependently (0.03–3mg/kg and 0.3–30mg/kg, respectively) decreased the serum intact parathyroid hormone (iPTH) and serum Ca2+ levels; however, the effect of upacicalcet on the reduction in serum Ca2+ disappeared at extracellular Ca2+ levels below the physiologically range, even when administered at a dose higher (100-fold) than the effective dose. Furthermore, upacicalcet did not affect gastric emptying in normal rats when administered up to a dose of 10mg/kg (300-fold higher than the dose affecting serum iPTH levels), while the administration of cinacalcet significantly slowed gastric emptying by approximately 50%. These findings suggest that upacicalcet has potential as an alternative calcimimetic agent with good pharmacological properties and a lower risk of hypocalcemia and gastrointestinal complications.

Mathematical modeling of calcium homeostasis in female rats: An analysis of sex differences and maternal adaptations

Calcium plays a vital role in various biological processes, including muscle contractions, blood clotting, skeletal mineralization, and cell signaling. While extracellular calcium makes up less than 1% of total body calcium, it is tightly regulated since too high or too low extracellular calcium concentration can have dangerous effects on the body. Mathematical modeling is a well-suited approach to investigate the complex physiological processes involved in calcium regulation. While mathematical models have been developed to study calcium homeostasis in male rats, none have been used to investigate known sex differences in hormone levels nor the unique physiological states of pregnancy and lactation. Calcitriol, the active form of vitamin D, plays a key role in intestinal calcium absorption, renal calcium reabsorption, and bone remodeling. It has been shown that, when compared to age-matched male rats, females have significantly lower calcitriol levels. In this study we first seek to investigate the impact of this difference as well as other known sex differences on calcium homeostasis using mathematical modeling. Female bodies differ from male bodies in that during their lifetime they may undergo massive adaptations during pregnancy and lactation. Indeed, maternal adaptations impact calcium regulation in all mammals. In pregnant rodents, intestinal absorption of calcium is massively increased in the mother’s body to meet the needs of the developing fetus. In a lactating rodent, much of the calcium needs of milk are met by bone resorption, intestinal absorption, and renal calcium reabsorption. Given these observations, the goal of this project is to develop multi-scale whole-body models of calcium homeostasis that represents (1) how sex differences impact calcium homeostasis in female vs. male rats and (2) how a female body adapts to support the excess demands brought on by pregnancy and lactation. We used these models to quantify the impact of individual sex differences as well as maternal adaptations during pregnancy and lactation. Additionally, we conducted “what if” simulations to test whether sex differences in calcium regulation may enable females to better undergo maternal adaptations required in pregnancy and lactation than males.

Reproducibility of drug-induced effects on the contractility of an engineered heart tissue derived from human pluripotent stem cells.

Introduction: Engineered heart tissues (EHTs) are three-dimensional culture platforms with cardiomyocytes differentiated from human pluripotent stem cells (hPSCs) and were designed for assaying cardiac contractility. For drug development applications, EHTs must have a stable function and provide reproducible results. We investigated these properties with EHTs made with different tissue casting batches and lines of differentiated hPSC-cardiomyocytes and analyzed them at different times after being fabricated. Methods: A video-optical assay was used for measuring EHT contractile outputs, and these results were compared with results from motion traction analysis of beating hPSC-cardiomyocytes cultured as monolayers in two-dimensional cultures. The reproducibility of induced contractile variations was tested using compounds with known mechanistic cardiac effects (isoproterenol, EMD-57033, omecamtiv mecarbil, verapamil, ranolazine, and mavacamten), or known to be clinically cardiotoxic (doxorubicin, sunitinib). These drug-induced variations were characterized at different electrical pacing rates and variations in intracellular calcium transients were also assessed in EHTs. Results: To ensure reproducibility in experiments, we established EHT quality control criteria based on excitation-contraction coupling and contractile sensitivity to extracellular calcium concentration. In summary, a baseline contractile force of 0.2mN and excitation-contraction coupling of EHTs were used as quality control criteria to select suitable EHTs for analysis. Overall, drug-induced contractile responses were similar between monolayers and EHTs, where a close relationship was observed between contractile output and calcium kinetics. Contractile variations at multiple time points after adding cardiotoxic compounds were also detectable in EHTs. Discussion: Reproducibility of drug-induced effects in EHTs between experiments and relative to published work on these cellular models was generally observed. Future applications for EHTs may require additional mechanistic criteria related to drug effects and cardiac functional outputs to be measured in regard to specific contexts of use.

Purinergic Activation of Store-Operated Calcium Entry (SOCE) Regulates Cell Migration in Metastatic Ovarian Cancer Cells.

Store-operated calcium entry (SOCE) is an important process in calcium signaling. Its role in physiological and pathological events is well recognized. However, in cancerous systems, the importance of SOCE in relation to the degree of cancer aggressiveness, as well as its regulation by ligands such as purinergic molecules, are not well documented. This study aimed to characterize a differential effect of the P2Y2 receptor (promoted by UTP of 10 µM and inhibited by ARC118925XX of 1 µM) on intracellular calcium response between metastatic (SKOV-3) and non-metastatic (CAOV-3) ovarian cell lines in conditions of normal (1.5 mM) and zero extracellular calcium concentration. The sustained calcium influx observed exclusively in SKOV-3 cells was associated with the presence of SOCE (promoted by thapsigargin (74.81 ± 0.94 ΔF) and sensitive to 2-APB (20.60 ± 0.85 ΔF)), whereas its absence in CAOV-3 cells (26.2 ± 6.1 ΔF) was correlated with a low expression of ORAI1. The relevance of SOCE in metastatic SKOV-3 cells was further corroborated when 2-APB significantly inhibited (40.4 ± 2.8% of covered area) UTP-induced cell migration (54.6 ± 3.7% of covered area). In conclusion, our data suggest that SOCE activation elicited by the P2Y2 receptor is involved in the aggressiveness of ovarian cancer cells.

Revealing Low Amplitude Signals of Neuroendocrine Cells through Disordered Silicon Nanowires-Based Microelectrode Array.

Today, the key methodology to study in vitro or in vivo electrical activity in a population of electrogenic cells, under physiological or pathological conditions, is by using microelectrode array (MEA). While significant efforts have been devoted to develop nanostructured MEAs for improving the electrophysiological investigation in neurons and cardiomyocytes, data on the recording of the electrical activity from neuroendocrine cells with MEA technology are scarce owing to their weaker electrical signals. Disordered silicon nanowires (SiNWs) for developing a MEA that, combined with a customized acquisition board, successfully capture the electrical signals generated by the corticotrope AtT-20cells as a function of the extracellular calcium (Ca2+ ) concentration are reported. The recorded signals show a shape that clearly resembles the action potential waveform by suggesting a natural membrane penetration of the SiNWs. Additionally, the generation of synchronous signals observed under high Ca2+ content indicates the occurrence of a collective behavior in the AtT-20cell population. This study extends the usefulness of MEA technology to the investigation of the electrical communication in cells of the pituitary gland, crucial in controlling several essential human functions, and provides new perspectives in recording with MEA the electrical activity of excitable cells.

Changes in intracellular calcium concentration level accompany age-related inhibitions of long-term potentiation in hippocampus induced by extremely low frequency electromagnetic fields.

Extremely low frequency electromagnetic fields (ELF-EMFs) have received increasing attention and in-depth research due to their ability to affect learning and memory functions. However, its regulation and intrinsic mechanism at different ages in early developmental stages remains unclear. In this article, the regulation of 15 Hz/2mT ELF-EMFs on long-term potentiation (LTP) persistence in the hippocampal CA1 region of Sprague-Dawley (SD) rats at early developmental stages (8-, 15-, 22-, and 29-day-old) are investigated by electrophysiological techniques. The results show that ELF-EMFs differentially inhibit LTP persistence due to age difference, and the younger the age, the more significant the inhibitory effect. Second, the inhibitory effect of ELF-EMFs on LTP persistence disappeared after the addition of 2-aminoethoxydiphenyl borate (2-APB) to block inositol-1,4,5-trisphosphate receptors (IP3 Rs) localized to intracellular calcium stores to reduce the intracellular calcium concentration ([Ca2+ ]i ), proving that the LTP persistence regulated by ELF-EMFs is associated with the IP3 Rs-mediated intracellular calcium stores. Finally, the level of [Ca2+ ]i was intervened by adjusting the extracellular calcium concentration([Ca2+ ]e ). Interestingly, the inhibitory effect of ELF-EMFs on LTP persistence in the 15-day-old group disappeared by increasing [Ca2+ ]e , whereas the inhibitory effect of ELF-EMFs on LTP persistence in the 29-day-old group appeared by decreasing [Ca2+ ]e . Our findings reveal the underlying mechanism of the effect of ELF-EMFs on synaptic plasticity in hippocampal CA1 area at early developmental stages and provide new insights into more rational application and protection of ELF-EMFs.

A novel high-throughput assay to produce ring-shaped 3D cardiac tissues from human induced pluripotent stem cells

Cardiac tissue engineering aims at creating contractile structures that can optimally reproduce the features of human cardiac tissue. These constructs, associated to the induced pluripotent stem cell (iPSC) technology, are becoming valuable tools to model some of the cardiac functions, to set preclinical platforms for drug testing, or to model cardiac pathologies. Nevertheless, the techniques used to generate engineered tissues still require expertise and produce a limited number of tissues per batch. To develop a novel assay which allows a fast, reproducible and high-throughput generation of 3D cardiac rings in a 96-well plate. A specific mold was designed to generate ring-shaped tissues around a central PEG pillar mounted on glass coverslips (Fig. 1). Cardiomyocytes derived from wild-type human iPSCs (hiPSC-CMs) were mixed with human dermal fibroblasts (HDF) and then seeded in the wells. An inhouse Matlab code was used to measure the deformation of the central pillar in time, deriving the force exerted by the tissue, and several contraction parameters. At day 14, the tissues were fixed and stained to evaluate their organization by confocal imaging. Finally, drug response of the tissues was assessed with different drugs. After seeding, the cells mix fell at the bottom of the PEG microwells and compacted around the pillars, forming 21 beating cardiac tissues per well. An iPSC-CM/fibroblast ratio of 3:1 was optimal to generate the highest number of stable tissues in time. The staining of vimentin and troponin T showed that the fibroblasts form a layer, stabilizing the iPSC-CMs which form a ring-shaped muscular fiber above. Tissue motion was monitored by real-time video-microscopy. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25 ± 0.55%, that maintained up to 14 days. The average stress developed was of 1,43 ± 0,37 μN/mm2. The cardiac constructs showed a positive inotropic response to increasing extracellular calcium concentrations and isoproterenol, while verapamil induced a strong negative inotropic response. Dofetilide induced arrhythmias at low concentrations and cardiotoxic effects. We report on the development on a novel, easy-to-use, reproducible and high-throughput platform to generate cardiac tissues using hiPSC-CMs. The platform can serve for disease modeling and pharmacological testing.

Inhibition of the calcium-sensing receptor by extracellular phosphate ions and by intracellular phosphorylation.

As both a sensor of extracellular calcium (Ca2+ o) concentration and a key controller of Ca2+ o homeostasis, one of the most interesting properties of the calcium-sensing receptor (CaR) is its sensitivity to, and modulation by, ions and small ligands other than Ca2+. There is emerging evidence that extracellular phosphate can act as a partial, non-competitive CaR antagonist to modulate parathyroid hormone (PTH) secretion, thus permitting the CaR to integrate mineral homeostasis more broadly. Interestingly, phosphorylation of certain intracellular CaR residues can also inhibit CaR responsiveness. Thus, negatively charged phosphate can decrease CaR activity both extracellularly (via association with arginine) and intracellularly (via covalent phosphorylation).

Extracellular Calcium-Induced Calcium Transient Regulating the Proliferation of Osteoblasts through Glycolysis Metabolism Pathways

During bone remodeling, high extracellular calcium levels accumulated around the resorbing bone tissue as soon as the activation of osteoclasts. However, if and how calcium is involved in the regulation of bone remodeling remains unclear. In this study, the effect of high extracellular calcium concentrations on osteoblast proliferation and differentiation, intracellular calcium ([Ca2+]i) levels, metabolomics, and the expression of proteins related to energy metabolism were investigated. Our results showed that high extracellular calcium levels initiated a [Ca2+]i transient via the calcium-sensing receptor (CaSR) and promoted the proliferation of MC3T3-E1 cells. Metabolomics analysis showed that the proliferation of MC3T3-E1 cells was dependent on aerobic glycolysis, but not the tricarboxylic acid cycle. Moreover, the proliferation and glycolysis of MC3T3-E1 cells were suppressed following the inhibition of AKT. These results indicate that calcium transient triggered by high extracellular calcium levels activated glycolysis via AKT-related signaling pathways and ultimately promoted the proliferation of osteoblasts.

Genomically anchored vitamin D receptor mediates an abundance of bioprotective actions elicited by its 1,25-dihydroxyvitamin D hormonal ligand.

The nuclear vitamin D receptor (VDR) mediates the actions of its physiologic 1,25-dihydroxyvitamin D3 (1,25D) ligand produced in kidney and at extrarenal sites during times of physiologic and cellular stress. The ligand-receptor complex transcriptionally controls genes encoding factors that regulate calcium and phosphate sensing/transport, bone remodeling, immune function, and nervous system maintenance. With the aid of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23), 1,25D/VDR primarily participates in an intricate network of feedback controls that govern extracellular calcium and phosphate concentrations, mainly influencing bone formation and mineralization, ectopic calcification, and indirectly supporting many fundamental roles of calcium. Beyond endocrine and intracrine effects, 1,25D/VDR signaling impacts multiple biochemical phenomena that potentially affect human health and disease, including autophagy, carcinogenesis, cell growth/differentiation, detoxification, metabolic homeostasis, and oxidative stress mitigation. Several health advantages conferred by 1,25D/VDR appear to be promulgated by induction of klotho, an anti-aging renal peptide hormone which functions as a co-receptor for FGF23 and, like 1,25D, regulates nrf2, foxo, mTOR and other cellular protective pathways. Among hundreds of genes for which expression is modulated by 1,25D/VDR either primarily or secondarily in a cell-specific manner, the resulting gene products (in addition to those expressed in the classic skeletal mineral regulatory tissues kidney, intestine, and bone), fall into multiple biochemical categories including apoptosis, cholesterol homeostasis, glycolysis, hypoxia, inflammation, p53 signaling, unfolded protein response and xenobiotic metabolism. Thus, 1,25D/VDR is a bone mineral control instrument that also signals the maintenance of multiple cellular processes in the face of environmental and genetic challenges.

Abstract 13: Myocardial Metabolic And Electrolyte Derangements Linked To PEA Rearrest

Background: Cardiac rearrest after return of spontaneous circulation (ROSC) is a significant barrier to successful resuscitation, making it a high priority for emergency care research. Regardless of initial arrest rhythm, rearrest is commonly due to pulseless electrical activity (PEA). However, mechanisms underlying PEA rearrest are poorly understood. Previously, myocardial contractile function during ischemia-reperfusion has been tied to alterations in extracellular sodium (Na+) and calcium (Ca2+) concentrations. Objective: To determine physiological predictors and explore potential mechanisms underlying susceptibility to PEA rearrest. Methods: Acute myocardial infarction (AMI) induced by left anterior descending artery (LAD) occlusion was followed by 8 min of VF, then pigs were resuscitated (defibrillation, CPR, epinephrine) to ROSC and the LAD was reperfused. Pigs were instrumented to measure metabolic variables from arterial and coronary sinus venous blood gases, electrolytes, and hemodynamics. Subjects that had PEA rearrest (n=7) were compared to those that did not rearrest (n=4). Results: Hemodynamic and primary arrest characteristics were similar between PEA and no rearrest groups, including ischemia duration, time to ROSC, CPR events, and epinephrine administration. At ROSC, hemodynamic and metabolic variables including pH, myocardial lactate consumption, myocardial arterial/venous oxygen difference were also similar between groups. Importantly, post-ROSC myocardial lactate consumption was significantly increased in the PEA group (3.9 vs. 0.5 mmol, p<0.03) as was coronary sinus Na+ (147 vs. 135 mEq/L, p<.03); however, coronary sinus Ca2+ was significantly reduced (1.9 vs. 2.3 mEq/L, p<.01 ). Taken together, these data suggest that metabolic substrate depletion may promote local electrolyte shifts contributing to mechanical dysfunction underlying PEA. Conclusion: PEA rearrest was associated with increased myocardial lactate consumption and derangements in cardiac Na+ and Ca2+ in a translational model with well controlled arrest characteristics. Improved understanding of mechanisms underlying post-ROSC electrolyte shifts may identify novel mechanisms and therapeutic approaches to prevent and treat PEA.