Changes In Ca2 Research Articles

Diethylcarbamazine elicits Ca2+ signals through TRP-2 channels that are potentiated by emodepside in Brugia malayi muscles.

Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis, which has serious effects on individuals' lives. Although current anthelmintics are effective at killing microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open transient receptor potential (TRP) channels in the muscles of adult female Brugia malayi, leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia, inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trial for the treatment of river blindness. Here, we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca2+ fluorescence in the muscle using Ca2+-imaging techniques. Diethylcarbamazine interacts with the transient receptor potential channel, C classification (TRPC) ortholog receptor TRP-2 to promote Ca2+ entry into the Brugia muscle cells, which can activate Slopoke (SLO-1) Ca2+-activated K+ channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca2+ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca2+ entry that is increased by emodepside activation of SLO-1 K+ channels.

Abstract 065: Reduction Of Endoplasmic Reticulum Stress By Treatment With Nifedipine In A Mouse Model Of Pre-eclampsia And In Human Placental Endothelial Cells

Introduction: Pre-eclampsia (PE) is characterized by high blood pressure (BP) and metabolic disorders during pregnancy, being the leading cause of maternal-neonatal mortality worldwide. The causes of PE are still unclear. We observed enhanced endoplasmic reticulum (ER) stress and increased cytoplasmic mobility of calreticulin, a Ca2+ ion binding chaperone protein, in placental endothelial cells (pECs) of mice with PE, indicating changes in Ca2+ mobilization. We aimed to describe the effect of nifedipine, a Ca2+ channel blocker, on ER stress in placentas of mice with PE and in human pECs exposed to hypoxia. Methods: We used heterozygous mice overexpressing human renin and angiotensinogen, developing high BP from gestational day (GD) 13.5, and controls (C57BL/6). Nifedipine (1 mg/kg in 0.5 ml saline, s.c.) or saline was administered at GD14.5 and placentas removed at GD18.5 (n=8-12/group). Protein expressions of IREα, ATF6 and PERK were evaluated by western blot. ECs were extracted from human placentas of healthy pregnancies by magnetic sorting and exposed to hypoxia (0.5% O2) or control conditions (8% O2) for 24h while treated with nifedipine (0.1 μM) or DMSO. Angiogenesis was assessed by tube formation assay on matrigel. Results were compared by 2-way ANOVA (mean±SD). Results: IREα expression was higher in placentas of PE mice treated with saline versus control (+306±64%, p=0.020) and significantly reduced with nifedipine versus saline in PE mice (-235±66%, p=0.009). Results were similar with PERK, which was increased in placentas of saline-treated PE mice versus saline controls (116±40%, p=0.037) and significantly reduced with nifedipine versus saline in PE mice (-141±38%, p=0.006). ATF6 was not different between groups, but was significantly reduced by nifedipine versus saline in PE mice (-76±24%, p=0.022). In pECs, nifedipine increased 17x the numbers of tubes formed on matrigel of cells exposed to hypoxia in comparison to DMSO control (p<0.001). Conclusion: The results describe an enhanced ER stress in placentas of PE mice and a positive effect of nifedipine on this stress. The pro-angiogenic effect of nifedipine was shown in pECs exposed to hypoxia. Further investigations are needed to confirm a potential angiogenic effect of nifedipine in PE.

Geochemical characteristics of natural springs within the Lenglongling fault zone related to the Menyuan Ms 6.9 earthquake on January 8, 2022, NW China

Geofluids from natural springs connect with the crust and/or mantle in many cases, and their geochemical anomalies could be significant for the study on faults activity and even earthquakes. Several natural springs are distributed along the Lenglongling fault zone (LLLFZ) in the northeastern margin of the Tibetan Plateau, where the Ms 6.9 Menyuan earthquake occurred on January 8th, 2022. Based on chemical and isotopic compositions (δD, δ18O, δ13C, and 3He/4He) of water and gas samples, the origin of geofluids and their potential correlation with fault activity even including earthquakes are preliminarily assessed in this paper. The δ13CCO2 values and 3He/4He ratios showed that the gas originating from the crust was associated with the metamorphism of carbonate rocks, whereas the δ18O and δD values of water samples indicated that the natural springs were predominantly infiltrated with precipitations from local mountains ranging 3.7 - 5.5 km in height. Obvious changes of Ca2+ and HCO3− concentrations in SZK spring waters in the surface rupture zones were observed in a short period (about three months) after the main shock, in contrast to those of the GSK springs far from the surface rupture zones. Such variations might be correlated with the stress increase prior to the 2022 Menyuan Ms 6.9 earthquake. The mechanical fracturing of surrounding limestone rocks during the slipping movement of LLLF could facilitate the water-rock interactions. Compared to three-month observations after the main shock, relatively higher concentrations of HCO3− and heavier δ18OH2O values of the LHG springs were also observed in the short-term period. The shallow stored formation water might be squeezed along the cracks and rose to the surface during earthquake tremors, causing a sandblasting water phenomenon.

Calcineurin stimulation by Cnb1p overproduction mitigates protein aggregation and α-synuclein toxicity in a yeast model of synucleinopathy

The calcium-responsive phosphatase, calcineurin, senses changes in Ca2+ concentrations in a calmodulin-dependent manner. Here we report that under non-stress conditions, inactivation of calcineurin signaling or deleting the calcineurin-dependent transcription factor CRZ1 triggered the formation of chaperone Hsp100p (Hsp104p)-associated protein aggregates in Saccharomyces cerevisiae. Furthermore, calcineurin inactivation aggravated α-Synuclein-related cytotoxicity. Conversely, elevated production of the calcineurin activator, Cnb1p, suppressed protein aggregation and cytotoxicity associated with the familial Parkinson’s disease-related mutant α-Synuclein A53T in a partly CRZ1-dependent manner. Activation of calcineurin boosted normal localization of both wild type and mutant α-synuclein to the plasma membrane, an intervention previously shown to mitigate α-synuclein toxicity in Parkinson’s disease models. The findings demonstrate that calcineurin signaling, and Ca2+ influx to the vacuole, limit protein quality control in non-stressed cells and may have implications for elucidating to which extent aberrant calcineurin signaling contributes to the progression of Parkinson’s disease(s) and other synucleinopathies.55zLzriqK_eYGg5W2i5yhiVideo

Abstract P2071: Mutation-Specific Calmodulin-Kinase II Activation In Hypertrophic Cardiomyopathy

Autophosphorylation of Calmodulin-Kinase IIδ (CaMKIIδ) at Thr-287 leads to autoactivation such that kinase activity is autonomous of Ca 2+ /CaM. In the heart, CaMKIIδ functions to regulate Ca 2+ homeostasis by targeting Ca 2+ handling proteins, including phospholamban (PLB). In sarcomeric hypertrophic cardiomyopathy (HCM), disruption of Ca 2+ homeostasis and aberrant autoactivation of CaMKIIδ has been linked to disease progression. We have previously shown that the pathogenic role of autonomous CaMKIIδ in HCM is mutation-specific. Transgenic mice expressing the HCM mutation R92W in cardiac troponin T (cTnT) exhibited an increase in autonomous CaMKIIδ while cTnT-R92L mice did not. The potentially clinically relevant mechanism(s) underlying this mutation specificity are unresolved. We hypothesize that a potential trigger of autonomous CaMKIIδ is accelerated myofilament Ca 2+ dissociation kinetics. We have previously shown that cTnT-R92W accelerates Ca 2+ dissociation, while cTnT-R92L decelerates Ca 2+ dissociation. Here, we extended our study to include cTnT-I79N and cTnT-R94H to determine if Ca 2+ dissociation rates can be linked to autonomous CaMKIIδ activity and thus provide a potential novel target in HCM. Our data show that cTnT-I79N accelerates Ca 2+ dissociation, and cTnT-R94H decelerates Ca 2+ dissociation, similar to our earlier findings. To determine whether the mutation-specific changes in Ca 2+ dissociation alter CaMKIIδ activity, transgenic mice expressing cTnT-R94H and cTnT-I79N were probed for autophosphorylated CaMKIIδ and Thr-17 phosphorylated PLB. At 2 months, PLB phosphorylation is unchanged in cTnT-R94H and cTnT-I79N mice. At 4 months, cTnT-I79N mice exhibit a trending increase in autonomous CaMKIIδ and cTnT-R94H mice exhibit a modest increase, similar to cTnT-R92W positive controls. These trends are matched by PLB phosphorylation levels. At 6 months, cTnT-R94H mice exhibit normal autonomous CaMKIIδ levels. While future and ongoing work will explore changes in myocellular Ca 2+ homeostasis in more depth, these preliminary data suggest that, unlike cTnT-R94H mice, cTnT-I79N mice exhibit elevated autonomous CaMKIIδ and these mutation-specific differences may underlie observed phenotypic variation in patients.

Abstract P3142: Engineering A Genetically Encoded Enhancer Of L-type Ca 2+ Currents

L-type calcium channels (LTCC) play a crucial role as they serve as vital portals for Ca 2+ entry into cardiac myocytes, neurons, and other physiological systems. Altered LTCC function is linked to various pathologies including cardiac arrhythmias and neurodevelopmental disorders. In particular, genetic studies have identified loss-of-function mutations in Ca V 1.2 channels in patients with shortened QT interval and Brugada Syndrome, as well as neuropsychiatric disorders. Given the broad physiological importance of LTCC in various tissues, conventional pharmacological agonists of LTCC may yield off-target effects and toxicity. Therefore, new approaches to upregulate LTCC function in a cell-type dependent manner is sought after. To do so, we here develop a genetically encoded enhancer of calcium channels (GeeCC, “geek”) by leveraging both a cardiac specific L-type channel modulator, leucine rich repeat containing protein 10 (Lrrc10), and selective nanobodies that target the LTCC complex. Specifically, Lrrc10 co-expression markedly upregulates LTCC currents. Furthermore, mutations in Lrrc10 are linked to cardiac diseases, including cardiomyopathies. Through extensive structure-function analysis, we identified a minimal action domain within Lrrc10 that is critical for functional modulation of LTCC. However, as this segment alone has a low affinity for the channel complex, its expression alone yielded minimal changes in Ca 2+ current amplitude. However, fusion of this Lrrc10 domain to a nanobody (nb.F3) that targets the Ca 2+ channel beta subunit resulted in a significant increase in the LTCC current amplitude. In depth analysis revealed exquisite selectivity of GeeCC for Ca V 1.2/Ca V 1.3 channels. In mouse cardiomyocytes, deployment of GeeCC via adenovirus enhances endogenous L-type currents when compared to uninfected controls or cardiomyocytes infected with nanobodies alone. Additionally, in neurons, co-expression of GeeCC enhanced excitation-transcription coupling. In all, we have developed a genetically-encoded approach to selectively enhance L-type currents. This strategy may enable new insights into cardiovascular and neuronal physiology and pathophysiology, and may have potential therapeutic applications.

Abstract P1121: Substrate Rigidity And Mybpc3 Genotype Regulate Development Of Hypertrophic Cardiomyopathy Structural And Functional Phenotypes In Human Ipsc-derived Micro-heart Muscle Arrays

Introduction: We hypothesized that mechanical loading is a key factor in the early stages of hypertrophic remodeling associated with pathogenesis of hypertrophic cardiomyopathy (HCM). We tested this hypothesis in vitro with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) bearing heterozygous loss-of-function mutations in Myosin Binding Protein C (MYBPC3). Cardiomyocytes were cultured in engineered micro-heart muscle arrays that were loaded by changing substrate rigidity. Methods: Isogenic control and MYBPC3 +/- hiPSC-CM were seeded into dogbone-shaped molds to form micro-heart muscle atop silicone rubber substrates with rigidity levels ranging from levels of embryonic (0.4 kPa) to fibrotic adult heart (114 kPa). Spontaneously contracting micro-tissues formed within 48 hours. 10 days after formation, tissues were monitored for action potentials (voltage sensitive dye, BeRST-1), Ca 2+ transients (GCaMP6f reporter), and contractility (substrate deflection). Cell and tissue architecture were assessed in 8 μm cryosections, and RNA transcription via qRT-PCR. Results: MYBPC3 +/- and control cardiomyocytes had similar physiology and morphology in 2D monolayers. However, MYBPC3 +/- tissues exhibited marked changes in Ca 2+ handling, which were exacerbated when substrate stiffness increased to the level of healthy heart muscle (15 kPa). This was concurrent with hypertrophy of MYBPC3 +/- cardiomyocytes at 15 kPa. MYBPC3 +/- tissue Ca 2+ handling abnormalities were linked to enhanced Ca 2+ intake rather than SERCA deficiency or excessive myofilament buffering. MYBPC3 +/- tissues exhibited mis-regulation of cardiac troponins on the RNA and protein localization levels, which may underlie the observed structural and physiological changes. Conclusions: Changes in the rigidity of the substrate, mimicking changes in heart muscle caused by blood pressure, pushed hiPSC-cardiac micro-tissues with MYBPC3 mutations to recapitulate key structural and electrophysiological characteristics of HCM. In contrast, control tissues exhibited hypertrophy and abnormal calcium handling, but only at extreme values of substrate stiffness. This suggests that HCM mutations may exaggerate load-dependent hypertrophic remodeling.

Calcium handling coupled to the endothelin ETA and ETB receptor-mediated vasoconstriction in resistance arteries: Differential regulation by PI3K, PKC and RhoK

Abnormal endothelin-1 (ET-1) activity is involved in the pathogenesis of vascular diseases such as essential and pulmonary arterial hypertension, coronary artery disease, and cerebrovascular disease, blockade of ET receptors having shown efficacy in clinical assays and experimental models of hypertension. Augmented Ca2+ influx and changes in Ca2+ sensitization associated with arterial vasoconstriction underlie increased systemic vascular resistance in hypertension. Since peripheral resistance arteries play a key role in blood pressure regulation, we aimed to determine here the specific Ca2+ signaling mechanisms linked to the ET receptor-mediated vasoconstriction in resistance arteries and their selective regulation by protein kinase C (PKC), Rho kinase (RhoK), the phosphatidylinositol 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK). ET-1-induced contraction was mediated by the endothelin ETA receptor with a minor contribution of vascular smooth muscle (VSM) endothelin ETB receptors. ET receptor activation elicited Ca2+ mobilization from intracellular stores, extracellular Ca2+ influx and Ca2+ sensitization associated with contraction in resistance arteries. Vasoconstriction induced by ET-1 was largely dependent on activation of canonical transient receptor potential channel 3 (TRPC3) and extracellular Ca2+ influx through nifedipine-sensitive voltage-dependent Ca2+ channels. PI3K inhibition reduced intracellular Ca2+ mobilization and Ca2+ entry without altering vasoconstriction elicited by ET-1, while PKC has dual opposite actions by enhancing Ca2+ influx associated with contraction, and by inhibiting Ca2+ release from intracellular stores. RhoK was a major determinant of the enhanced sensitivity of the contractile filaments underlying ET-1 vasoconstriction, with also a modulatory positive action on Ca2+ influx and intracellular Ca2+ release. Augmented RhoK and PKC activities are involved in vascular dysfunction in hypertension and vascular complications of insulin-resistant states, and these kinases are thus potential pharmacological targets in vascular diseases in which the ET pathway is impaired.

Exploring the acid neutralizing effect in rainwater collected at a tropical urban area: Central Valley, Costa Rica

We report on the chemical and the carbon isotopic composition of dissolved inorganic carbon (DIC) of rainwater collected between May and October 2020 in the Central Valley, Costa Rica. Precipitation samples were collected daily (N = 55) and analyzed for major ions, DIC, and δ13CDIC. Significant correlation (p < 0.05) between main acidic (SO42− and NO3−) and major alkaline (Ca2+ and NH4+) species confirmed a very effective acid neutralization effect in rainwater (average pH: 5.90 ± 0.74). Significant temporal variations (p < 0.05) of δ13CDIC indicated the contribution of carbonate salts in rainwater from May to October but also CO2 dissolution at the beginning of the wet season (May), probably due to increased CO2 emissions from soil degassing. Temporal changes of Ca2+ neutralization factors followed the observed changes in δ13CDIC, which confirmed the high buffer capacity of precipitation in our study. HYSPLIT analysis also revealed long-range contributions of pedogenetic carbonates (e.g., Saharan dust) responsible for the acid neutralization capacity of rainwater (e.g., from July to September). Principal component analysis showed that four main factors explain 65% of the variance are: i) acid neutralization processes (Ca2+ neutralization factor), ii) marine salts (Cl−, Na+), iii) fossil fuels (SO42−, NO3−), and iv) agriculture/fertilizers (NO3−, NH4+, K+). Our study demonstrated that a combined approach of chemical, isotope, and statistical analysis techniques can help unravel the mechanism of acid neutralization of rainwater in tropical urban areas. This information has strong implications for future studies related with the impact of acid deposition on ecosystem functioning, water quality, and infrastructure degradation.

CardIAP: calcium transients confocal image analysis tool.

One of the main topics of cardiovascular research is the study of calcium (Ca2+) handling, as even small changes in Ca2+ concentration can alter cell functionality (Bers, Annu Rev Physiol, 2014, 76, 107-127). Ionic calcium (Ca2+) plays the role of a second messenger in eukaryotic cells, associated with cellular functions such as cell cycle regulation, transport, motility, gene expression, and regulation. The use of fluorometric techniques in isolated cells loaded with Ca2+-sensitive fluorescent probes allows quantitative measurement of dynamic events occurring in living, functioning cells. The Cardiomyocytes Images Analyzer Python (CardIAP) application addresses the need to analyze and retrieve information from confocal microscopy images systematically, accurately, and rapidly. Here we present CardIAP, an open-source tool developed entirely in Python, freely available and useable in an interactive web application. In addition, CardIAP can be used as a standalone Python library and freely installed via PIP, making it easy to integrate into biomedical imaging pipelines. The images that can be generated in the study of the heart have the particularity of requiring both spatial and temporal analysis. CardIAP aims to open the field of cardiomyocytes and intact hearts image processing. The improvement in the extraction of information from the images will allow optimizing the usage of resources and animals. With CardIAP, users can run the analysis to both, the complete image, and portions of it in an easy way, and replicate it on a series of images. This analysis provides users with information on the spatial and temporal changes in calcium releases and characterizes them. The web application also allows users to extract calcium dynamics data in downloadable tables, simplifying the calculation of alternation and discordance indices and their classification. CardIAP aims to provide a tool that could assist biomedical researchers in studying the underlying mechanisms of anomalous calcium release phenomena.

Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta.

Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.

Hyperactivity of medial prefrontal cortex pyramidal neurons occurs in a mouse model of early-stage Alzheimer's disease without β-amyloid accumulation.

The normal function of the medial prefrontal cortex (mPFC) is essential for regulating neurocognition, but it is disrupted in the early stages of Alzheimer's disease (AD) before the accumulation of Aβ and the appearance of symptoms. Despite this, little is known about how the functional activity of medial prefrontal cortex pyramidal neurons changes as Alzheimer's disease progresses during aging. We used electrophysiological techniques (patch-clamping) to assess the functional activity of medial prefrontal cortex pyramidal neurons in the brain of 3xTg-Alzheimer's disease mice modeling early-stage Alzheimer's disease without Aβ accumulation. Our results indicate that firing rate and the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) were significantly increased in medial prefrontal cortex neurons from young Alzheimer's disease mice (4-5-month, equivalent of <30-year-old humans) compared to age-matched control mice. Blocking ionotropic glutamatergic NMDA receptors, which regulate neuronal excitability and Ca2+ homeostasis, abolished this neuronal hyperactivity. There were no changes in Ca2+ influx through the voltage-gated Ca2+ channels (VGCCs) or inhibitory postsynaptic activity in medial prefrontal cortex neurons from young Alzheimer's disease mice compared to controls. Additionally, acute exposure to Aβ42 potentiated medial prefrontal cortex neuronal hyperactivity in young Alzheimer's disease mice but had no effects on controls. These findings indicate that the hyperactivity of medial prefrontal cortex pyramidal neurons at early-stage Alzheimer's disease is induced by an abnormal increase in presynaptic glutamate release and postsynaptic NMDA receptor activity, which initiates neuronal Ca2+ dyshomeostasis. Additionally, because accumulated Aβ forms unconventional but functional Ca2+ channels in medial prefrontal cortex neurons in the late stage of Alzheimer's disease, our study also suggests an exacerbated Ca2+ dyshomeostasis in medial prefrontal cortex pyramidal neurons following overactivation of such VGCCs.

Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes

Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging.TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found.In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 are expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.

Physiology of intracellular calcium buffering.

Calcium signaling underlies much of physiology. Almost all the Ca2+ in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels in most cells. Physiological Ca2+ buffers include small molecules and proteins, and experimentally Ca2+ indicators will also buffer calcium. The chemistry of interactions between Ca2+ and buffers determines the extent and speed of Ca2+ binding. The physiological effects of Ca2+ buffers are determined by the kinetics with which they bind Ca2+ and their mobility within the cell. The degree of buffering depends on factors such as the affinity for Ca2+, the Ca2+ concentration, and whether Ca2+ ions bind cooperatively. Buffering affects both the amplitude and time course of cytoplasmic Ca2+ signals as well as changes of Ca2+ concentration in organelles. It can also facilitate Ca2+ diffusion inside the cell. Ca2+ buffering affects synaptic transmission, muscle contraction, Ca2+ transport across epithelia, and the killing of bacteria. Saturation of buffers leads to synaptic facilitation and tetanic contraction in skeletal muscle and may play a role in inotropy in the heart. This review focuses on the link between buffer chemistry and function and how Ca2+ buffering affects normal physiology and the consequences of changes in disease. As well as summarizing what is known, we point out the many areas where further work is required.

High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate in the reticular Ca2+ leak towards mitochondria.

Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure μM or sub-μM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked.

Curvature Matters: Modeling Calcium Binding to Neutral and Anionic Phospholipid Bilayers.

In this work, the influence of membrane curvature on the Ca2+ binding to phospholipid bilayers is investigated by means of molecular dynamics simulations. In particular, we compared Ca2+ binding to flat, elastically buckled, or uniformly bent zwitterionic and anionic phospholipid bilayers. We demonstrate that Ca2+ ions bind preferably to the concave membrane surfaces in both types of bilayers. We also show that the membrane curvature leads to pronounced changes in Ca2+ binding including differences in free ion concentrations, lipid coordination distributions, and the patterns of ion binding to different chemical groups of lipids. Moreover, these effects differ substantially for the concave and convex membrane monolayers. Comparison between force fields with either full or scaled charges indicates that charge scaling results in reduction of the Ca2+ binding to curved phosphatidylserine bilayers, while for phosphatidylcholine membranes, calcium binds only weakly for both force fields.

PO-01-209 THE MECHANISMS OF IMPAIRED CALCIUM HANDLING UNDERLYING THE INCREASED SUSCEPTIBILITY TO ATRIAL FIBRILLATION IN METABOLIC SYNDROME

Atrial fibrillation (AF) is becoming increasingly common partly due to the prevalence of metabolic syndrome (MetS). Altered Ca2+-handling proteins on the atrial cellular membrane as well as across the sarcoplasmic reticulum (SR) network and axial tubules due to this condition can precipitate AF through elevation of Ca2+ in junctional clefts, triggering Ca2+ sparks. Through the stochastic recruitment of these sparks, a Ca2+ wave can manifest to activate neighbouring ryanodine receptors (RyRs), stimulating Na+/Ca2+ exchangers (INaCa) into potentially triggering arrhythmogenic spontaneous action potentials (SAPs). However, the mechanisms by which MetS can disrupt Ca2+ handling and trigger SAPs is not well understood. To investigate the mechanisms of impaired calcium handling underlying the increased susceptibility to AF in MetS. We utilized a 3D model of subcellular Ca2+ signalling by Zhang et al. 2022, which compromises of a network of Ca2+-release units (CRUs) and ion channel and exchanger formulations from the Grandi et al. common pool model. The Zhang et al. model far more accurately captures the processes by which Ca2+ sparks can occur and terminate through the inclusion of crucial membrane ion channels/transporters within the extensive network of axial tubules interwoven across the CRUs (Fig A). Based on experimental studies, MetS meditated functional change in Ca2+-handling proteins was replicated by increasing the conductivity of SERCA and RyR and reducing ICaL and INaCa. To evaluate the contribution of each individual remodeled Ca2+-handling component to the membrane potential and Ca2+ sparks, a series of simulations was conducted where we first incorporated each individual ionic remodeling into the control model, then from the MetS model, we reversed each remodeled Ca2+-handling component separately. We have demonstrated that SAPs were attributed to a decrease in INaCa and an increase in SERCA. For the former, this also corresponded to a much greater incidence of Ca2+ sparks compared to other cases. However, for the latter, increasing SERCA surprisingly had a negligible impact on the genesis of these sparks (Fig B). This suggests that an alternate pathway can potentially exist by which upregulated SERCA function can induce SAPs. Increased Ca2+ spark incidence due to functional changes in Ca2+-handling proteins is not the sole mechanism to SAP genesis. Our study therefore highlights that MetS-mediated AF can occur through different pathways.

New Findings: Hindlimb Unloading Causes Nucleocytoplasmic Ca2+ Overload and DNA Damage in Skeletal Muscle.

Disuse atrophy of skeletal muscle is associated with a severe imbalance in cellular Ca2+ homeostasis and marked increase in nuclear apoptosis. Nuclear Ca2+ is involved in the regulation of cellular Ca2+ homeostasis. However, it remains unclear whether nuclear Ca2+ levels change under skeletal muscle disuse conditions, and whether changes in nuclear Ca2+ levels are associated with nuclear apoptosis. In this study, changes in Ca2+ levels, Ca2+ transporters, and regulatory factors in the nucleus of hindlimb unloaded rat soleus muscle were examined to investigate the effects of disuse on nuclear Ca2+ homeostasis and apoptosis. Results showed that, after hindlimb unloading, the nuclear envelope Ca2+ levels ([Ca2+]NE) and nucleocytoplasmic Ca2+ levels ([Ca2+]NC) increased by 78% (p < 0.01) and 106% (p < 0.01), respectively. The levels of Ca2+-ATPase type 2 (Ca2+-ATPase2), Ryanodine receptor 1 (RyR1), Inositol 1,4,5-tetrakisphosphate receptor 1 (IP3R1), Cyclic ADP ribose hydrolase (CD38) and Inositol 1,4,5-tetrakisphosphate (IP3) increased by 470% (p < 0.001), 94% (p < 0.05), 170% (p < 0.001), 640% (p < 0.001) and 12% (p < 0.05), respectively, and the levels of Na+/Ca2+ exchanger 3 (NCX3), Ca2+/calmodulin dependent protein kinase II (CaMK II) and Protein kinase A (PKA) decreased by 54% (p < 0.001), 33% (p < 0.05) and 5% (p > 0.05), respectively. In addition, DNase X is mainly localized in the myonucleus and its activity is elevated after hindlimb unloading. Overall, our results suggest that enhanced Ca2+ uptake from cytoplasm is involved in the increase in [Ca2+]NE after hindlimb unloading. Moreover, the increase in [Ca2+]NC is attributed to increased Ca2+ release into nucleocytoplasm and weakened Ca2+ uptake from nucleocytoplasm. DNase X is activated due to elevated [Ca2+]NC, leading to DNA fragmentation in myonucleus, ultimately initiating myonuclear apoptosis. Nucleocytoplasmic Ca2+ overload may contribute to the increased incidence of myonuclear apoptosis in disused skeletal muscle.

3D-assembled microneedle ion sensor-based wearable system for the transdermal monitoring of physiological ion fluctuations

Monitoring human health is of considerable significance in biomedicine. In particular, the ion concentrations in blood are important reference indicators related to many diseases. Microneedle array-based sensors have enabled promising breakthroughs in continuous health monitoring due to their minimally invasive nature. In this study, we developed a microneedle sensing-array integrated system to continuously detect subcutaneous ions to monitor human health status in real time based on a fabrication strategy for assembling planar microneedle sheets to form 3D microneedle arrays. The limitations of preparing 3D microneedle structures with multiple electrode channels were addressed by assembling planar microneedle sheets fabricated via laser micromachining; the challenges of modifying closely spaced microneedle tips into different functionalized types of electrodes were avoided. The microneedle sensing system was sufficiently sensitive for detecting real-time changes in Ca2+, K+, and Na+ concentrations, and it exhibited good detection performance. The in vivo results showed that the ion-sensing microneedle array successfully monitored the fluctuations in Ca2+, K+, and Na+ in the interstitial fluids of rats in real time. By using an integrated circuit design, we constructed the proposed microneedle sensor into a wearable integrated monitoring system. The integrated system could potentially provide information feedback for diseases related to physiological ion changes.

Transcriptional Basis of Ca2+ Remodeling Reversal Induced by Polyamine Synthesis Inhibition in Colorectal Cancer Cells.

Colorectal cancer (CRC) is associated with mutations in APC/Wnt leading to c-myc activation and the overexpression of ODC1, the limiting step in polyamine synthesis. CRC cells also display a remodeling of intracellular Ca2+ homeostasis that contributes to cancer hallmarks. As polyamines may modulate Ca2+ homeostasis during epithelial tissue repair, we investigated whether polyamine synthesis inhibition may reverse Ca2+ remodeling in CRC cells and, if so, the molecular basis for this reversal. To this end, we used calcium imaging and transcriptomic analysis in normal and CRC cells treated with DFMO, an ODC1 suicide inhibitor. We found that polyamine synthesis inhibition partially reversed changes in Ca2+ homeostasis associated with CRC, including a decrease in resting Ca2+ and SOCE along with an increased Ca2+ store content. We also found that polyamine synthesis inhibition reversed transcriptomic changes in CRC cells without affecting normal cells. Specifically, DFMO treatment enhanced the transcription of SOCE modulators CRACR2A; ORMDL3; and SEPTINS 6, 7, 8, 9, and 11, whereas it decreased SPCA2, involved in store-independent Orai1 activation. Therefore, DFMO treatment probably decreased store-independent Ca2+ entry and enhanced SOCE control. Conversely, DFMO treatment decreased the transcription of the TRP channels TRPC1 and 5, TRPV6, and TRPP1 while increasing TRPP2, thus probably decreasing Ca2+ entry through TRP channels. Finally, DFMO treatment enhanced the transcription of the PMCA4 Ca2+ pump and mitochondrial channels MCU and VDAC3 for enhanced Ca2+ extrusion through the plasma membrane and mitochondria. Collectively, these findings suggested the critical role of polyamines in Ca2+ remodeling in colorectal cancer.