Ca Signaling Research Articles

Plant traits regulated metal(loid)s in dominant herbs in an antimony mining area of the karst zone, China.

Understanding how plant functional traits respond to mining activities and impact metal(loid)saccumulation in dominant species is crucial for exploring the driving mechanisms behind plant community succession and predicting the ecological restoration potential of these plants. In this study, we investigated four dominant herbaceous species (Artemisia argyi, Miscanthus sinensis, Ficus tikoua, and Ageratina adenophora) growing on antimony (Sb) mining sites (MS) with high Sb and arsenic (As) levels, as well as non-mining sites (NMS). The aim was to analyze the variations in functional traits and their contribution to Sb and As concentrations in plants. Our results indicate that mining activities enhanced soil nitrogen (N) limitation and phosphorus (P) enrichment, while significantly reducing the plant height of three species, except for F. tikoua. The four species absorbed more calcium (Ca) to ensure higher tolerance to Sb and As levels, which is related to the activation of Ca signaling pathways and defense mechanisms. Furthermore, plant Sb and As concentrations were dependent on soil metal(loid) levels and plant element stoichiometry. Overall, these findings highlight the regulatory role of plant element traits in metal(loid) concentrations, warranting widespread attention and further study in the future.

#1447 Calcimimetics improve impaired cardiac function and contractility in chronic phosphate loading by activating the CaSR/cAMP signaling cascade

Abstract Background and Aims High phosphate loading stimulate the synthesis of PTH and FGF23 and is associated with increased cardiovascular (CV) mortality. In hemodialysis patients, the administration of calcimimetics led to reductions in PTH and FGF23 and the latter was associated with a lower rate of CV events. We have previously shown that mice with a high dietary phosphate load show elevated PTH and FGF23 levels and develop impaired systolic cardiac function, which could be improved by calcimimetics. Calcimimetics are allosteric activators of the calcium-sensing receptor (CaSR). Phosphate (Pi) was shown to inactivate the parathyroid CaSR at concentrations >1.4 mM Pi. Whether calcimimetics contribute to CV event reduction indirectly via lowering PTH and FGF23 or directly via activation of the cardiac CaSR is unknown. Method Wild-type mice were fed a high phosphate diet (HPD) ± etelcalcetide (Etl) or a normal phosphate diet (NPD), cardiac function was assessed by echocardiography and Millar catheterization, and cardiac tissue was harvested for histology and RNAseq analyses. In addition, isolated adult mouse cardiomyocytes (AMCM) were stimulated ex vivo with PTH, FGF23 or Pi, contractility and calcium handling were measured with the addition of Etl and activity analyses were performed. Results In addition to the positive effect on HPD-induced systolic cardiac dysfunction, Etl improved impaired LV contractility and reduced fibrosis. RNAseq analyses revealed reduced Ca, PKA and cAMP signaling by HPD. Ex vivo stimulation of AMCM with PTH and FGF23 had no functional effects. In contrast, Pi significantly worsened contractility and Ca handling, which was blunted by Etl co-stimulation. Downstream of cardiac CaSR, Pi reduced the formation of cAMP, which led to PKA-dependent dephosphorylation of PLN and subsequently to lower Serca2a activity, which was ultimately responsible for the reduced contractility. Etl led to improved cardiomyocyte contractility via increased cAMP synthesis and increased PLN phosphorylation and Serca2a activity. Conclusion Our data suggest that Etl ameliorates the Pi-induced pathologic cardiac phenotype associated with high phosphate load via direct activation of CaSR/cAMP signaling cascade, resulting in improvement of cardiomyocyte contractility.

Modelling spatio-temporal interactions between second messengers Ca and cAMP in a pancreatic -cell.

Calcium serves as a widespread second messenger in almost every human and animal cell. The regulation of various cellular processes, such as transcriptional control and the kinetics of membrane channels, is significantly influenced by intracellular calcium ions (Ca ), and linkages between Ca and other second messengers should activate signaling networks. The passage of ions across the cell membrane regulates Ca levels in pancreatic -cells and requires the coordinated interaction of various ion transport mechanisms and organelles. The signaling of Ca in -cells and its interactions with the intracellular dynamics of cyclic adenosine monophosphate (cAMP) is poorly understood. Therefore, the current investigation proposes a mathematical model to illustrate the spatiotemporal dynamical interaction between Ca and cAMP. In order to construct a one-dimensional mathematical model, the fundamental initial and boundary conditions derived from the physiological characteristics of the -cell are incorporated. The numerical results were obtained by MATLAB simulations using the finite element method and the Crank-Nicolson method. The current study aims to offer an update on regulation between Ca and cAMP signaling circuits, with a focus on interactions that occur in localized areas of the -cell. The model gives the individual effect of each parameter on the regulation of Ca and cAMP profiles in a -cell. Evidently, impairments in the regulation of messenger pathways contribute to the pathological conditions, as demonstrated by the results obtained.

Effect of increased CO2 on calcium homeostasis and signaling in a marine diatom

AbstractOcean acidification influences photosynthesis, respiration, and metabolism in marine diatoms, leading to changes in diatom growth performance and shifts in phytoplankton communities. Previous studies have demonstrated that increases in seawater CO2 concentrations affect the uptake of trace metals such as iron, zinc, copper, and cobalt by marine diatoms. However, the influence of increased CO2 on calcium, which plays a vital role as a secondary messenger in various signaling pathways within organisms, has received limited attention so far. This study examined the effect of increased CO2 on Ca homeostasis and signaling in the marine diatom Phaeodactylum tricornutum. While seawater acidification had little effect on the diatom's growth, it significantly changed cell properties (surface topography, adhesion, and surface potential). Elevated CO2 concentrations reduced calcium accumulation P. tricornutum and lowered the rise of cytosolic Ca2+ transients stimulated by toxic aldehyde, phosphorus supplement, and hypo‐osmotic stress. Our results suggest that a continuous rise in atmospheric CO2 may alter diatoms' response to environmental cues.

Modulating mitochondrial phosphate transport shapes intracellular Ca signaling to impact arrhythmogenesis

Modulating mitochondrial phosphate transport shapes intracellular Ca signaling to impact arrhythmogenesis

Role of Mitochondrial ROS for Calcium Alternans in Atrial Myocytes.

Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternations in CaT amplitude and is causally linked to atrial fibrillation (AF). Mitochondria play a significant role in cardiac excitation-contraction coupling and Ca signaling through redox environment regulation. In isolated rabbit atrial myocytes, ROS production is enhanced during CaT alternans, measured by fluorescence microscopy. Exogenous ROS (tert-butyl hydroperoxide) enhanced CaT alternans, whereas ROS scavengers (dithiothreitol, MnTBAP, quercetin, tempol) alleviated CaT alternans. While the inhibition of cellular NADPH oxidases had no effect on CaT alternans, interference with mitochondrial ROS (ROSm) production had profound effects: (1) the superoxide dismutase mimetic MitoTempo diminished CaT alternans and shifted the pacing threshold to higher frequencies; (2) the inhibition of cyt c peroxidase by SS-31, and inhibitors of ROSm production by complexes of the electron transport chain S1QEL1.1 and S3QEL2, decreased the severity of CaT alternans; however (3) the impairment of mitochondrial antioxidant defense by the inhibition of nicotinamide nucleotide transhydrogenase with NBD-Cl and thioredoxin reductase-2 with auranofin enhanced CaT alternans. Our results suggest that intact mitochondrial antioxidant defense provides crucial protection against pro-arrhythmic CaT alternans. Thus, modulating the mitochondrial redox state represents a potential therapeutic approach for alternans-associated arrhythmias, including AF.

Calcium signal regulated carbohydrate metabolism in wheat seedlings under salinity stress.

This study aimed to explore the mechanism by which calcium (Ca) signal regulated carbohydrate metabolism and exogenous Ca alleviated salinity toxicity. Wheat seedlings were treated with sodium chloride (NaCl, 150mM) alone or combined with 500μM calcium chloride (CaCl2), lanthanum chloride (LaCl3) and/or ethylene glycol tetraacetic acid (EGTA) to primarily analyse carbohydrate starch and sucrose metabolism, as well as Ca signaling components. Treatment with NaCl, EGTA, or LaCl3 alone retarded wheat-seedling growth and decreased starch content accompanied by weakened ribulose-1,5-bisphosphate carboxylation/oxygenase (Rubisco) and Rubisco activase activities, as well as enhanced glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, alpha-amylase, and beta-amylase activities. However, it increased the sucrose level, up-regulated the sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities and TaSPS and TaSuSy expression together, but down-regulated the acid invertase (SA-Inv) and alkaline/neutral invertase (A/N-Inv) activities and TaSA-Inv and TaA/N-Inv expression. Except for unchanged A/N-Inv activities and TaA/N-Inv expression, adding CaCl2 effectively blocked the sodium salt-induced changes of these parameters, which was partially eliminated by EGTA or LaCl3 presence. Furthermore, NaCl treatment also significantly inhibited Ca-dependent protein kinases and Ca2+-ATPase activities and their gene expression in wheat leaves, which was effectively relieved by adding CaCl2. Taken together, CaCl2 application effectively alleviated the sodium salt-induced retardation of wheat-seedling growth by enhancing starch anabolism and sucrose catabolism, and intracellular Ca signal regulated the enzyme activities and gene expression of starch and sucrose metabolism in the leaves of sodium salt-stressed wheat seedlings.

Modulation of calcium signaling and metabolic pathways in endothelial cells with magnetic fields.

Calcium signaling plays a crucial role in various physiological processes, including muscle contraction, cell division, and neurotransmitter release. Dysregulation of calcium levels and signaling has been linked to a range of pathological conditions such as neurodegenerative disorders, cardiovascular disease, and cancer. Here, we propose a theoretical model that predicts the modulation of calcium ion channel activity and calcium signaling in the endothelium through the application of either a time-varying or static gradient magnetic field (MF). This modulation is achieved by exerting magnetic forces or torques on either biogenic or non-biogenic magnetic nanoparticles that are bound to endothelial cell membranes. Since calcium signaling in endothelial cells induces neuromodulation and influences blood flow control, treatment with a magnetic field shows promise for regulating neurovascular coupling and treating vascular dysfunctions associated with aging and neurodegenerative disorders. Furthermore, magnetic treatment can enable control over the decoding of Ca signals, ultimately impacting protein synthesis. The ability to modulate calcium wave frequencies using MFs and the MF-controlled decoding of Ca signaling present promising avenues for treating diseases characterized by calcium dysregulation.

Sequentialremoval of cation/H+ exchangers reveals their additive role in elemental distribution, calcium depletionand anoxia tolerance.

Multiple Arabidopsis H+ /Cation exchangers (CAXs) participate in high-capacity transport into the vacuole. Previous studies have analysed single and double mutants that marginally reduced transport; however, assessing phenotypes caused by transport loss has proven enigmatic. Here, we generated quadruple mutants (cax1-4: qKO) that exhibited growth inhibition, an 85% reduction in tonoplast-localised H+ /Ca transport, and enhanced tolerance to anoxic conditions compared to CAX1 mutants. Leveraging inductively coupled plasma mass spectrometry (ICP-MS)and synchrotron X-ray fluorescence (SXRF), we demonstrate CAX transporters work together to regulate leaf elemental content: ICP-MS analysis showed that the elemental concentrations in leaves strongly correlated with the number of CAX mutations; SXRF imaging showed changes in element partitioning not present in single CAX mutants and qKO had a 40% reduction in calcium (Ca) abundance. Reduced endogenous Ca may promote anoxia tolerance; wild-type plants grown in Ca-limited conditions were anoxia tolerant. Sequential reduction of CAXs increased mRNA expression and protein abundance changes associated with reactive oxygen species and stress signalling pathways. Multiple CAXs participate in postanoxia recovery as their concerted removal heightened changes in postanoxia Ca signalling. This work showcases the integrated and diverse function of H+ /Cation transporters and demonstrates the ability to improve anoxia tolerance through diminishing endogenous Ca levels.

Elementary intracellular Ca signals approximated as a transition of release channel system from a metastable state.

Cardiac muscle contraction is initiated by an elementary Ca signal (called Ca spark) which is achieved by collective action of Ca release channels in a cluster. The mechanism of this synchronization remains uncertain. We approached Ca spark activation as an emergent phenomenon of an interactive system of release channels. We constructed a weakly lumped Markov chain that applies an Ising model formalism to such release channel clusters and probable open channel configurations and demonstrated that spark activation is described as a system transition from a metastable to an absorbing state, analogous to the pressure required to overcome surface tension in bubble formation. This yielded quantitative estimates of the spark generation probability as a function of various system parameters. We performed numerical simulations to find spark probabilities as a function of sarcoplasmic reticulum Ca concentration, obtaining similar values for spark activation threshold as our analytic model, as well as those reported in experimental studies. Our parametric sensitivity analyses also showed that the spark activation threshold decreased as Ca sensitivity of RyR activation and RyR cluster size increased.

Dynamics of laser-induced plasma and cavitation bubble at high pressures and the impacts on underwater LIBS signals

Characteristics of laser-induced plasma depends strongly on its ambient pressure, and a deeper understanding of the high pressure impact on the underwater plasma emission is essential for the application of LIBS in the deep-sea. In this work, we investigated the temporal evolution of underwater plasma and cavitation bubble at the high pressures up to 50 MPa, by using fast imaging and shadowgraph techniques. It showed that as the ambient pressure increases, the plasma expansion is much suppressed with a stronger emission intensity and a slower emission decay, but it quenches much earlier and ends with a sharp drop at the higher pressures. From the shadowgraph imaging results, it showed that the increase in pressure has a great impact on the bubble evolution. Both the bubble maximum radius and the bubble lifetime decrease dramatically with the pressure as a consequence of faster dynamics. By comparing between the plasma and bubble results, we demonstrated the critical role of bubble dynamics on the characterization of plasma emission at high pressures. At relatively low pressures, the bubble expansion time is long enough to finish the plasma radiation. While at high pressures, strong bubble-plasma interaction occurs that will guide the plasma evolution behavior at the late stage. The plasma can gain energy from the confinement effect caused by the bubble shrinking process which leads to an increase in the emission intensity, but it will quench immediately after the bubble collapse. Such effects caused by bubble dynamics at high pressures explain well the evolution behaviors of underwater LIBS signals of OH, Ca II, Ca I, and CaOH, considering the different lifetimes of these emitting species in the plasma. This work reveals the complex impacts of high pressure on underwater plasma emissions and underwater LIBS signals.

Physiological responses of plants to in vivo X-ray damage from X-ray fluorescence measurements: insights from anatomical, elemental, histochemical, and ultrastructural analyses.

X-ray fluorescence spectroscopy (XRF) is a powerful technique for the in vivo assessment of plant tissues. However, the potential X-ray exposure damages might affect the structure and elemental composition of living plant tissues, leading to artefacts in the recorded data. Herein, we exposed in vivo soybean (Glycine max (L.) Merrill) leaves to several X-ray doses through a polychromatic benchtop microprobe X-ray fluorescence spectrometer, modulating the photon flux density by adjusting either the beam size, current, or exposure time. Changes in the irradiated plant tissues' structure, ultrastructure, and physiology were investigated through light and transmission electron microscopy (TEM). Depending on X-ray exposure dose, decreased K and X-ray scattering intensities and increased Ca, P, and Mn signals on soybean leaves were recorded. Anatomical analysis indicated the necrosis of epidermal and mesophyll cells on the irradiated spots, where TEM images revealed the collapse of cytoplasm and cell wall breaking. Furthermore, the histochemical analysis detected the production of reactive oxygen species and the inhibition of chlorophyll autofluorescence in these areas. Under certain X-ray exposure conditions, e.g. high photon flux density and long exposure time, XRF measurements may affect the soybean leaves structures, elemental composition, and cellular ultrastructure, inducing programmed cell death. Our characterization shed light on the plant's responses to the X-ray-induced radiation damage and might help to establish proper X-ray radiation limits and novel strategies for in vivo benchtop-XRF analysis of vegetal materials.

Stimulating Role of Calcium and Cyclic GMP in Mediating the Effect of Magnetopriming for Alleviation of Salt Stress in Soybean Seedlings

This current study examined the role of calcium (Ca) and Cyclic GMP (cGMP) in mitigating the adverse effect of salt stress through magnetopriming of soybean cultivar JS-335 seeds with a static magnetic field (SMF, 200 mT for 1 h). The salt stress (50 mMNaCl) extensively reduced the early seedling growth (64%), vigour Index-I (71%), vigour Index-II (39%), total amylase (59%), protease (63%), and nitrate reductase (NR, 19%) activities in un-primed soybean seedlings. However, magnetopriming and Ca treatment enhanced all of these measured parameters along with remarkable increase in reactive oxygen species (ROS) and nitric oxide (NO) content. The exogenous application of Ca2+, cGMP and ROS regulators such as nifedipine (Ca2+ channel blocker), EGTA, ethylene glycol-β-amino ethyl ether tetra acetic acid (Ca2+chelators), genistein (cGMP blocker), and dimethyl thiourea (DMTU, H2O2 inhibitor) negatively affects the SMF-induced seedling length, seedling vigour, ROS, NO, and enzyme activities such as protease, total amylase, and NR in soybean seedlings. Results presented by using specific various biochemical inhibitors of Ca, cGMP, or ROS signalling in vivo indicated that Ca and cGMP are also involved with ROS and NO in the signal transduction of magnetic field enthused soybean seed germination and seedling growth under salt stress.

Orai1 is an Entotic Ca2+ Channel for Non-Apoptotic Cell Death, Entosis in Cancer Development.

Entosis is a non-apoptotic cell death process that forms characteristic cell-in-cell structures in cancers, killing invading cells. Intracellular Ca2+ dynamics are essential for cellular processes, including actomyosin contractility, migration, and autophagy. However, the significance of Ca2+ and Ca2+ channels participating in entosis is unclear. Here, it is shown that intracellular Ca2+ signaling regulates entosis via SEPTIN-Orai1-Ca2+ /CaM-MLCK-actomyosin axis. Intracellular Ca2+ oscillations in entotic cells show spatiotemporal variations during engulfment, mediated by Orai1 Ca2+ channels in plasma membranes. SEPTIN controlled polarized distribution of Orai1 for local MLCK activation, resulting in MLC phosphorylation and actomyosin contraction, leads to internalization of invasive cells. Ca2+ chelators and SEPTIN, Orai1, and MLCK inhibitors suppress entosis. This study identifies potential targets for treating entosis-associated tumors, showing that Orai1 is an entotic Ca2+ channel that provides essential Ca2+ signaling and sheds light on the molecular mechanism underlying entosis that involves SEPTIN filaments, Orai1, and MLCK.

Exogenous calcium regulates the growth and development of Pinus massoniana detecting by physiological, proteomic, and calcium-related genes expression analysis

Pinus massoniana is an important industrial crop tree species commonly used for timber and wood pulp for papermaking, rosin, and turpentine. This study investigated the effects of exogenous calcium (Ca) on P. massoniana seedling growth, development, and various biological processes and revealed the underlying molecular mechanisms. The results showed that Ca deficiency led to severe inhibition of seedling growth and development, whereas adequate exogenous Ca markedly improved growth and development. Many physiological processes were regulated by exogenous Ca. The underlying mechanisms involved diverse Ca-influenced biological processes and metabolic pathways. Calcium deficiency inhibited or impaired these pathways and processes, whereas sufficient exogenous Ca improved and benefited these cellular events by regulating several related enzymes and proteins. High levels of exogenous Ca facilitated photosynthesis and material metabolism. Adequate exogenous Ca supply relieved oxidative stress that occurred at low Ca levels. Enhanced cell wall formation, consolidation, and cell division also played a role in exogenous Ca-improved P. massoniana seedling growth and development. Calcium ion homeostasis and Ca signal transduction-related gene expression were also activated at high exogenous Ca levels. Our study facilitates the elucidation of the potential regulatory role of Ca in P. massoniana physiology and biology and is of guiding significance in Pinaceae plant forestry.

Modulating mitochondrial phosphate transport shapes intracellular Ca signaling to impact arrhythmogenesis.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondria Ca uptake and buffering. Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knock out (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. An inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Our results suggest that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce Ca waves and cardiac arrhythmias in CPVT.

Heart failure (HF) alters distribution and spatial association profiles of calmodulin (CaM) and CaM target protein mRNAs.

Aberrant Ca-CaM signaling has been implicated in various congenital and acquired cardiac pathologies, including arrhythmia, hypertrophy, and HF. We examined the impact of HF induced by trans-aortic constriction (TAC) on the distribution of the three CaM mRNAs (Calm 1,2 and 3) and their key protein target mRNAs (Ryr2, Scn5a, Camk2d, NOS1 and Cacna1c) in cardiomyocytes, using fluorescence in situ hybridization (RNAScope™). HF resulted in specific changes in the pattern of localization of Calms, manifested in redistribution of Calm3 from the cell periphery towards the perinuclear area and enhanced Calm2 attraction to the perinuclear area compared to sham myocytes. Additionally, HF resulted in redistribution of mRNAs for certain CaM target mRNAs. Particularly, NOS1 localization shifted from the cell periphery towards the perinuclear area, Cacna1c, Camk2d and Scn5a abundance increased at the perinuclear area, and Ryr2 attracted even closer to the cell periphery in HF myocytes compared to sham myocytes. The strength of non-random attraction/repulsion was measured as the maximal deviation between the observed distribution of nearest neighbor distances from the distribution predicted under complete spatial randomness. Consistent with the observed alterations in abundance and distribution of CaM and CaM target mRNAs, HF resulted in increased attraction between Calm1 and Scn5a, Ryr2 and Camk2d, between Calm2 and Ryr2 and Camk2d; and between Calm3 and NOS1 and Scn5a. In contrast, the attraction between Calm3 and Ryr2 decreased in HF myocytes compared to sham. Collectively, these results suggest distribution of Calms and their association with key target protein mRNAs undergo substantial alterations in heart failure. These results have new important implications for organization of Ca signaling in normal and diseased heart.

The role of mitochondrial-associated-membranes in Ca-dependent arrhythmias.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Recent studies suggest that mitochondria function as a protective Ca buffer to absorb RyR2-mediated aberrant Ca release in CPVT. However, the molecular mechanism underlying the protective Ca buffering function of CPVT mitochondria remains unclear. We hypothesize that the tethering between SR and mitochondria, also known as mitochondria-associated-membranes (MAMs) are promoted in CPVT to facilitate SR-mitochondria Ca transfer. Moreover, manipulating SR-mitochondria tethering or MAMs impacts arrhythmogenesis in CPVT. Ventricular myocytes were isolated from a CPVT model of CASQ2 knockout (Cnull) mouse. Cellular immunofluorescence assays and western blots were employed to detect MAMs remodeling in CPVT cells. Pharmacological and genetic approaches were employed to disrupt or promote MAMs and examine its effect on cellular arrhythmogenesis using live-cell imaging. SR-mitochondria tethering was assessed by examining the interaction between RyR2 and Voltage-dependent anion channel (VDAC), a protein localized in the mitochondrial-outer-membrane. As compared with wild-type (WT), we detected a higher degree of colocalization of immunofluorescence between RyR2 and VDAC in Cnull cells, and increased RyR2-VDAC interactions. The expression of SR-mitochondria tethering protein Mitofusin2 was increased in MAMs isolated from CPVT hearts. When treated with colchicine to induce a partial disruption of MAMs, arrhythmogenic Ca waves were exacerbated in intact Cnull cells. In permeabilized cells, colchicine also increased the frequency of Ca waves and reduced mitochondrial Ca uptake. Manipulation of MAMs by genetically targeting Mitofusin 2 also affected the frequency of Ca waves. Our results suggest that SR-mitochondria tethering or MAMs shape intracellular Ca signaling to impact arrhythmogenesis. Targeting this signaling microdomain may represent a novel strategy to reduce Ca waves and Ca-dependent arrhythmias.

CNS-focused high throughput screening for RyR-targeted therapeutics.

Using high-throughput screening (HTS) assays based on fluorescence lifetime (FLT)-detected FRET, we have identified compounds that allosterically modulate ryanodine receptor (RyR) calcium release channels, to prevent or reduce the elevated Ca2+ leak that fuels age-related neurodegeneration. RyRs are responsible for Ca release from intracellular storage organelles (endoplasmic reticulum – ER; sarcoplasmic reticulum – SR), and the resulting Ca signaling is essential for many cellular processes. RyRs have been intensively studied for decades in SR vesicles purified from skeletal muscle (RyR1) or heart (RyR2), where dysregulated RyR function – particularly a leaky state of the resting RyR – has been identified as a targetable culprit to interrupt the vicious cycle that compromises cell physiology in age-related pathologies. Recently RyR2 dysfunction in neurons has been implicated as a major contributor to Alzheimer's disease. Our FRET-based HTS detects the RyR leaky state by monitoring binding of the accessory proteins calmodulin, FKBP12.6, and DPc10. Under conditions that mimic a pathological state, we have carried out HTS of the 50k-compound CNS library (ChemBridge), to identify modulators of RyR2 in native SR membranes. This screen yielded 603 compounds (Hits) that reproducibly altered FRET between screens. With therapeutic feasibility a priority, freshly prepared stock solutions of 85 of the most promising compounds, validated for purity and chemical structure, were then evaluated for their dose-response in FRET and functional assays measuring 3[H]ryanodine binding. The resulting Hit compounds comprised seven unique chemical clusters that were either RyR isoform-specific or altered both RyR1 and RyR2 activity. Structure-activity relationship studies are ongoing. Good candidate compounds for treatment of neurodegeneration will progress to testing on RyR function in neurons from AD mouse models. This work is supported by NIH grant R43AG069582 (JJT/RLC).

Evidence for the role of sound on the growth and signal response in duckweed

ABSTRACT Sound vibration, an external mechanical force, has been proven to modulate plant growth and development like rain, wind, and vibration. However, the role of sound on plants, especially on signal response, has been usually neglected in research. Herein, we investigated the growth state, gene expression, and signal response in duckweed treated with soft music. The protein content in duckweed after music treatment for 7 days was about 1.6 times that in duckweed without music treatment. Additionally, the potential maximum photochemical efficiency of photosystem II (Fv/Fm) ratio in duckweed treated with music was 0.78, which was significantly higher in comparison with the control group (P < .01). Interestingly, music promoted the Glu and Ca signaling response. To further explore the global molecular mechanism, we performed transcriptome analysis and the library preparations were sequenced on an Illumina Hiseq platform. A total of 1296 differentially expressed genes (DEGs) were found for all these investigated genes in duckweed treated with music compared to the control group. Among these, up-regulation of the expression of metabolism-related genes related to glycolysis, cell wall biosynthesis, oxidative phosphorylation, and pentose phosphate pathways were found. Overall, these results provided a molecular basis to music-triggered signal response, transcriptomic, and growth changes in duckweed, which also highlighted the potential of music as an environmentally friendly stimulus to promote improved protein production in duckweed.