Amplitude Of Ca2 Research Articles

Effects of Varying Transverse and Axial Tubules in a Three-Dimensional Model of Calcium Signaling in the Human Atrial Myocyte

T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in ventricular myocytes. Atrial myocytes are generally thought to possess sparse irregular transverse-tubular (TT) components, as opposed to the highly dense and regular ventricular TT system. Axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum that include ryanodine receptor (RyR) clusters, characterized by rapid activation of Ca2+-induced Ca2+ release, have also been identified in atrial myocytes. AT and TT remodeling and changes in distribution, composition, and phosphorylation status of Ca2+ release units are thought to underlie Ca2+ abnormalities in atrial fibrillation (AF), the most common cardiac arrhythmia. Here, we performed a computational analysis to investigate how changes in the tubular network affect human atrial electrophysiology. We modified our well-established three-dimensional model of Ca2+ signaling in the rabbit ventricular myocyte to develop an analogous model in the human atrium. We also coupled the Ca2+ signaling model to our well-established model of membrane electrophysiology in the human atrial myocyte. We systematically varied TT and AT density and RyR distribution and assessed the effect on Ca2+ spark and wave properties. When TT density is low, as shown in isolated atrial myocytes, the model recapitulates the typical U-shaped Ca2+ wave seen experimentally in transverse confocal line-scan images. Introduction of ATs resulted in W-shaped Ca2+ waves, reflecting more synchronous Ca2+ release. The frequency and amplitude of Ca2+ release events were affected by both density of TT and AT, as well as RyR distribution. Our newly developed three-dimensional model of the human atrial myocyte will be employed to investigate whether and how AF-induced cellular electrical and structural remodeling effects collectively contribute to the membrane potential and Ca2+ abnormalities seen in AF.

Coupling of Calcium- and Membrane Clocks Ignites De Novo Spontaneous Action Potential in Dormant Guinea Pig Sinoatrial Nodal Cells via Camp-PKA Signaling

Firing rate and rhythm of spontaneous action potentials (APs) in sinoatrial nodal cells (SANC) are controlled via the fidelity of clock coupling: rhythmic, spontaneous, local Ca2+ releases (LCRs) that emerges beneath the cell membrane (‘Ca2+ clock’) during diastole couples to sarcolemmal electrogenic mechanisms (‘membrane clock’). We hypothesized that dormant guinea pig SANC, i.e. cells that don’t fire spontaneous APs, represent an extreme form of clock uncoupling, and increased cAMP-mediated, PKA-dependent phosphorylation of the coupled-clock system proteins ignite rhythmic AP firing in dormant SANC. Membrane potential of dormant GP SANC averaged −38±1mV(n=46), ICaL, If, and IK current densities were comparable to those that fire spontaneous APs, and LCRs were small and random. Isoproterenol or CPT-cAMP led to de novo spontaneous AP generation in ∼50% of dormant SANC. The initial response involved increases in LCR size and rhythmicity and small amplitude, spontaneous APs and whole-cell Ca2+ transients. Average LCR size continued to increase, average LCR period shortened, partially synchronizing LCRs to late diastole, paralleled by MDP hyperpolarization to ∼-60 mV. AP cycle length shortened similar to LCR period. β-AR stimulation increased availability of ICaL and IK, and If. Inhibition of Ca2+ clock by CPA precluded the isoproterenol-induced AP generation in 91% of dormant SANC, but If block by ivabradine did not inhibit the activation. Ivabradine, however, prolonged the transition between dormant and steady firing state following the initiation of β-AR stimulation. When β-AR stimulation/CPT-cAMP was removed, all changes reversed in order and SANC again became dormant. At least in some SANC, dormancy represents membrane & Ca2+ clock uncoupling that is rescued via cAMP-mediated, PKA-induced increased function of both membrane and Ca2+ clock proteins.

Температура модулирует инотропный эффект бета2-адренорецепторов через изменение продукции NO в изолированных левых предсердиях мыши

Stimulation of beta-adrenergic receptors (ARs) in response to sympathetic system activation increases heart contractility and rate. The beta1- and 2-ARs are main subtypes expressed in the cardiomyocytes. In the present work, using recording contractions and optical detection of calcium transient and nitric oxide levels, we have studied effects of the beta2-AR stimulation at different temperatures (25 and 35°С) in mice left atria. We found that increase in temperature from 25 to 35°C leads to an attenuation of the positive inotropic effect of beta2-AR stimulation which is accompanied by a decrease in amplitude of Ca2+ transient and an increase in NO synthesis. Under these conditions, pharmacological inhibition of NO production causes recovery of the effects of beta2-AR activation on the contractility and Ca2+ transient. Experiments using hydroxycholesterol interfering with coupling beta2-AR to NO synthesis also confirmed the sensitivity of this link to the temperature. We suggest that temperature plays a role in the coupling between beta2-AR and NO-dependent signaling that could be important for cardiac adaptation.

Altered Ca2+ and Na+ Homeostasis in Human Hypertrophic Cardiomyopathy: Implications for Arrhythmogenesis.

Hypertrophic cardiomyopathy (HCM) is the most common mendelian heart disease, with a prevalence of 1/500. HCM is a primary cause of sudden death, due to an heightened risk of ventricular tachyarrhythmias that often occur in young asymptomatic patients. HCM can slowly progress toward heart failure, either with preserved or reduced ejection fraction, due to worsening of diastolic function. Accumulation of intra-myocardial fibrosis and replacement scars underlies heart failure progression and represents a substrate for sustained arrhythmias in end-stage patients. However, arrhythmias and mechanical abnormalities may occur in hearts with little or no fibrosis, prompting toward functional pathomechanisms. By studying viable cardiomyocytes and trabeculae isolated from inter-ventricular septum samples of non-failing HCM patients with symptomatic obstruction who underwent myectomy operations, we identified that specific abnormalities of intracellular Ca2+ handling are associated with increased cellular arrhytmogenesis and diastolic dysfunction. In HCM cardiomyocytes, diastolic Ca2+ concentration is increased both in the cytosol and in the sarcoplasmic reticulum and the rate of Ca2+ transient decay is slower, while the amplitude of Ca2+-release is preserved. Ca2+ overload is the consequence of an increased Ca2+ entry via L-type Ca2+-current [due to prolongation the action potential (AP) plateau], combined with a reduced rate of Ca2+-extrusion through the Na+/Ca2+ exchanger [due to increased cytosolic (Na+)] and a lower expression of SERCA. Increased late Na+ current (INaL) plays a major role, as it causes both AP prolongation and Na+ overload. Intracellular Ca2+ overload determines an higher frequency of Ca2+ waves leading to delayed-afterdepolarizations (DADs) and premature contractions, but is also linked with the increased diastolic tension and slower relaxation of HCM myocardium. Sustained increase of intracellular [Ca2+] goes hand-in-hand with the increased activation of Ca2+/calmodulin-dependent protein-kinase-II (CaMKII) and augmented phosphorylation of its targets, including Ca2+ handling proteins. In transgenic HCM mouse models, we found that Ca2+ overload, CaMKII and increased INaL drive myocardial remodeling since the earliest stages of disease and underlie the development of hypertrophy, diastolic dysfunction and the arrhythmogenic substrate. In conclusion, diastolic dysfunction and arrhythmogenesis in human HCM myocardium are driven by functional alterations at cellular and molecular level that may be targets of innovative therapies.

PO-137 Epigenetic regulation of exercise-improved LTCC and BKCa channels function in hypertension mesenteric arteries

Objective To investigate the epigenetic mechanism of the voltage-gated L-type Ca2+ channel (LTCC) and the large-conductance Ca2+-activated K+ channel (BKCa) function in mesenteric arterial myocytes improved by regular aerobic exercise in hypertension.
 Methods 12-week-old male SHR and WKY rats were randomly assigned to sedentary and exercise training groups, respectively. Exercise groups were performed a moderate-intensity treadmill running. After 8 weeks, patch clamp study, Ca2+ image, Western blot, qPCR, bisulfite sequencing PCR were used to detect the LTCC and BKCa channel currents, BKCa single channel gating properties, Ca2+ spark, mRNA and protein expression of LTCC α1c together with BKCa α and β1 subunits, DNA methylation level of α1c and β1 gene promoter region, miR-328 expression. In vitro experiment，miR-328 mimic and miR-328 inhibitor were transfected into cultured arterial myocytes to make miR-328 overexpressing or silencing, the mRNA and protein expression of α1c subunits were determined after 48 h transfection.
 Results 1) After 8 weeks of exercise, SBP in both exercise groups of WKY and SHR were significantly lower than that of their sedentary counterparts. 2) Exercise normalized the increased LTCC and BKCa current density of mesenteric arterial myocytes in SHR. 3) Exercise attenuated the increased single BKCa channel open Probability (Po) and the amplitude of Ca2+ spark in hypertension. 4) Exercise inhibited the upregulated mRNA and protein expression of BKCa β1 subunit in mesenteric arteries from SHR; β1 gene promoter was demethylation in hypertension, exercise increased the methylation level at β1 gene promoter of SHR. 5) The protein expression of LTCC α1c subunit was significantly increased in SHR, while decreased by exercise; the expression of miR-328 in mesenteric arteries was highly negative correlation with α1c subunit. 6) The miR-328 overexpression by transfecting miR-328 mimic decreased α1c subunit protein level significantly, while miR-328 inhibitor made α1c subunit a slight increase.
 Conclusions Regular aerobic exercise efficiently reduces blood pressure of SHR, enhances β1 gene promoter methylation, mediates miR-328 inhibiting the α1c expression at post-transcriptional level, which might be the epigenetic mechanism underlying exercise-improved LTCC and BKCa channels function in mesenteric arteries of hypertension.

Anoctamin 5/TMEM16E facilitates muscle precursor cell fusion.

Limb-girdle muscular dystrophy type 2L (LGMD2L) is a myopathy arising from mutations in ANO5; however, information about the contribution of ANO5 to muscle physiology is lacking. To explain the role of ANO5 in LGMD2L, we previously hypothesized that ANO5-mediated phospholipid scrambling facilitates cell-cell fusion of mononucleated muscle progenitor cells (MPCs), which is required for muscle repair. Here, we show that heterologous overexpression of ANO5 confers Ca2+-dependent phospholipid scrambling to HEK-293 cells and that scrambling is associated with the simultaneous development of a nonselective ionic current. MPCs isolated from adult Ano5 -/- mice exhibit defective cell fusion in culture and produce muscle fibers with significantly fewer nuclei compared with controls. This defective fusion is associated with a decrease of Ca2+-dependent phosphatidylserine exposure on the surface of Ano5 -/- MPCs and a decrease in the amplitude of Ca2+-dependent outwardly rectifying ionic currents. Viral introduction of ANO5 in Ano5 -/- MPCs restores MPC fusion competence, ANO5-dependent phospholipid scrambling, and Ca2+-dependent outwardly rectifying ionic currents. ANO5-rescued MPCs produce myotubes having numbers of nuclei similar to wild-type controls. These data suggest that ANO5-mediated phospholipid scrambling or ionic currents play an important role in muscle repair.

Calcium-handling abnormalities underlying atrial arrhythmogenesis in a Fontan operation canine model.

Atrial tachyarrhythmia (AT) is a common complication in patients who have undergone a Fontan operation. In this study, we investigated whether abnormal Ca2+ handling contributes to the Fontan operation-related atrial arrhythmogenic substrate. Mongrel dogs were randomly assigned to sham and Fontan groups. The Fontan operation model was developed by performing an atriopulmonary anastomosis. After 14days, an electrophysiological study was performed to evaluate the AT vulnerability. Ca2+ handlingproperties were measured by loading atrial cardiomyocytes (CMs) with fura-2 AM. The L-type Ca2+ (ICa-L) and Na+-Ca2+exchanger (INCX) currentsof the CMs were recorded by the whole-cell patch-clamp technique. The key Ca2+ handling proteins expression was assessed by Western blotting. The AT inducibility was higher in the Fontan group than in the sham group (85.71 vs. 14.29%, P < 0.05). The Fontan operation resulted in decreased Ca2+ transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca2+ content, but in enhanced diastolic intracellular Ca2+ concentration and SR Ca2+ leak in the atrial CMs. The spontaneous CaT events, triggered ectopic activity and INCX density were increased, but ICa-L density was reduced in CMs from the Fontan atria (all P < 0.05). Additionally, the Fontan operation resulted in decreased SR Ca2+ ATPase expression and Cav1.2 expression, but in increased NCX1 and Ser2814-phosphorylated ryanodine receptor 2. The calmodulin-dependent protein kinase II expression and function were markedly enhanced in the Fontan atria. The Fontan operation caused atrial CM Ca2+ handling abnormalities that produced arrhythmogenic-triggered activity and increased vulnerability to AT in experimental Fontan dogs.

Mutation in the Sip1 transcription factor leads to a disturbance of the preconditioning of AMPA receptors by episodes of hypoxia in neurons of the cerebral cortex due to changes in their activity and subunit composition. The protective effects of interleukin-10

The Sip1 mutation plays the main role in pathogenesis of the Mowat-Wilson syndrome, which is characterized by the pronounced epileptic symptoms. Cortical neurons of homozygous mice with Sip1 mutation are resistant to AMPA receptor activators. Disturbances of the excitatory signaling components are also observed on such a phenomenon of neuroplasticity as hypoxic preconditioning. In this work, the mechanisms of loss of the AMPA receptor's ability to precondition by episodes of short-term hypoxia were investigated on cortical neurons derived from the Sip1 homozygous mice. The preconditioning effect was estimated by the level of suppression of the AMPA receptors activity with hypoxia episodes. Using fluorescence microscopy, we have shown that cortical neurons from the Sip1fl/fl mice are characterized by the absence of hypoxic preconditioning effect, whereas the amplitude of Ca2+-responses to the application of the AMPA receptor agonist, 5-Fluorowillardiine, in neurons from the Sip1 mice brainstem is suppressed by brief episodes of hypoxia. The mechanism responsible for this process is hypoxia-induced desensitization of the AMPA receptors, which is absent in the cortex neurons possessing the Sip1 mutation. However, the appearance of preconditioning in these neurons can be induced by phosphoinositide-3-kinase activation with a selective activator or an anti-inflammatory cytokine interleukin-10.

The Interplay of Rogue and Clustered Ryanodine Receptors Regulates Ca2+ Waves in Cardiac Myocytes.

Ca2+ waves in cardiac myocytes can lead to arrhythmias owing to delayed after-depolarisations. Based on Ca2+ regulation from the junctional sarcoplasmic reticulum (JSR), a mathematical model was developed to investigate the interplay of clustered and rogue RyRs on Ca2+ waves. The model successfully reproduces Ca2+ waves in cardiac myocytes, which are in agreement with experimental results. A new wave propagation mode of “spark-diffusion-quark-spark” is put forward. It is found that rogue RyRs greatly increase the initiation of Ca2+ sparks, further contribute to the formation and propagation of Ca2+ waves when the free Ca2+ concentration in JSR lumen ([Ca2+]lumen) is higher than a threshold value of 0.7 mM. Computational results show an exponential increase in the velocity of Ca2+ waves with [Ca2+]lumen. In addition, more CRUs of rogue RyRs and Ca2+ release from rogue RyRs result in higher velocity and amplitude of Ca2+ waves. Distance between CRUs significantly affects the velocity of Ca2+ waves, but not the amplitude. This work could improve understanding the mechanism of Ca2+ waves in cardiac myocytes.

Norepinephrine Increases Cytosolic Ca2+ in Neurons of the Paraventricular Nucleus of the Hypothalamus

The paraventricular nucleus (PVN), an important nucleus of the hypothalamus, is involved in cardiorespiratory and central autonomic responses to a variety of stimuli or stressors. The PVN receives dense innervation from catecholaminergic neurons in the nucleus tractus solitarii, ventrolateral medulla, and locus coeruleus. Stressors, including hypoxia, activate these projections to the PVN, releasing neurotransmitters and modulators including norepinephrine (NE). Within the PVN, adrenergic receptor activation by NE increases excitatory postsynaptic currents, and induces depolarization and elevated action potential discharge. However, NE's role in modulating cytosolic Ca2+ in PVN neurons remains to be determined. In this study, we determined the effects of NE on PVN neuron cytosolic Ca2+ using fura‐2 ratiometric fluorescent Ca2+ imaging. PVN neurons were dissociated from 3–4 wk old male Sprague‐Dawley rats and used within 12 hours of isolation. Neurons were exposed to aCSF baseline (Bsl, 5 min), followed by vehicle (5 min) or NE (100 mM, 5 min). Compared to aCSF Bsl, PVN neurons showed stable basal Ca2+ during vehicle controls (n=34). In contrast, addition of 100 mM NE elevated basal Ca2+ (n=69). To examine the contribution of Ca2+ from internal sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) stores in this elevation, neurons were exposed to the SERCA blocker Thapsigargin (TG, 1 mM, 30 min). When NE was added in the presence of TG the increase in basal Ca2+ persisted, although slightly attenuated (n=11). To examine if NE elevates cytosolic Ca2+ via extracellular Ca2+ entry, cells were exposed to aCSF with zero Ca2+. In the absence of extracellular Ca2+, NE did not elevate basal Ca2+ and rather showed a significant decrease (n=63). Spontaneous Ca2+ peaks in PVN neurons were also recorded. While NE did not affect the number of spontaneous peaks or the number of cells that exhibited spontaneous peaks, NE did increase spontaneous peak amplitude (n=69). NE in presence of TG replicated NE's increase in spontaneous peak amplitude (n=11). NE in the absence of extracellular Ca2+ significantly decreased spontaneous peak amplitude (n=63). To study the potential influence of NE on use (voltage)‐dependent Ca2+ channels, cells were repeatedly depolarized with 55 mM K+ (5×, 20 sec, 5 min intervals). NE or vehicle was applied between the 3rd and 4th depolarization and the amplitude of Ca2+ peaks and basal Ca2+ was evaluated. Across the 5 peaks, there was a slight decrease in amplitude over time that was not altered by vehicle (n=34). Application of 100 mM NE induced a small, non‐significant increase in Ca2+ peak amplitude, and a significant increase in basal Ca2+ (n=34). In contrast to the NE response that occurred in neurons studied immediately after isolation, PVN neurons incubated for 2–5 days saw the effects of NE greatly diminished, if not completely abolished, and 24 hour incubation in 10 mM NE did not restore NE sensitivity. These preliminary findings demonstrate NE increases cytosolic Ca2+, likely from an influx of extracellular Ca2+, and support previous work that NE is associated with depolarization of PVN neurons.Support or Funding InformationHL 098602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Variations in Ca2+ Influx Can Alter Chelator-Based Estimates of Ca2+ Channel-Synaptic Vesicle Coupling Distance.

The timing and probability of synaptic vesicle fusion from presynaptic terminals is governed by the distance between voltage-gated Ca2+ channels (VGCCs) and Ca2+ sensors for exocytosis. This VGCC-sensor coupling distance can be determined from the fractional block of vesicular release by exogenous Ca2+ chelators, which depends on biophysical factors that have not been thoroughly explored. Using numerical simulations of Ca2+ reaction and diffusion, as well as vesicular release, we examined the contributions of conductance, density, and open duration of VGCCs, and the influence of endogenous Ca2+ buffers on the inhibition of exocytosis by EGTA. We found that estimates of coupling distance are critically influenced by the duration and amplitude of Ca2+ influx at active zones, but relatively insensitive to variations of mobile endogenous buffer. High concentrations of EGTA strongly inhibit vesicular release in close proximity (20-30 nm) to VGCCs if the flux duration is brief, but have little influence for longer flux durations that saturate the Ca2+ sensor. Therefore, the diversity in presynaptic action potential duration is sufficient to alter EGTA inhibition, resulting in errors potentially as large as 300% if Ca2+ entry durations are not considered when estimating VGCC-sensor coupling distances.SIGNIFICANT STATEMENT The coupling distance between voltage-gated Ca2+ channels and Ca2+ sensors for exocytosis critically determines the timing and probability of neurotransmitter release. Perfusion of presynaptic terminals with the exogenous Ca2+ chelator EGTA has been widely used for both qualitative and quantitative estimates of this distance. However, other presynaptic terminal parameters such as the amplitude and duration of Ca2+ entry can also influence EGTA inhibition of exocytosis, thus confounding conclusions based on EGTA alone. Here, we performed reaction-diffusion simulations of Ca2+-driven synaptic vesicle fusion, which delineate the critical parameters influencing an accurate prediction of coupling distance. Our study provides guidelines for characterizing and understanding how variability in coupling distance across chemical synapses could be estimated accurately.

Expression and regulation of M-type K+ channel in PC12 cells and rat adrenal medullary cells.

M-type K+ channels contribute to the resting membrane potential in the sympathetic ganglion neurons of various animals, whereas their expression in adrenal medullary (AM) cells has been controversial. The present experiment aims to explore the expression of M channels comprising the KCNQ2 subunit in the rat AM cell and its immortalized cell line PC12 cells at the protein level and how its expression in PC12 cells is regulated. The KCNQ2 isoform was recognized in homogenates of PC12 cells but not the rat adrenal medullae by immunoblotting and KCNQ2-like immunoreactivity (IR) was detected in PC12 cells but not in rat AM cells. When the PC12 cells were maintained in a dexamethasone-containing medium, KCNQ2-like IR in the cells was suppressed, whereas the removal of fetal bovine serum from the culture medium for 1day resulted in an increase in KCNQ2-like IR. A similar enhancement occurred when PC12 cells were cultured under conditions where glucocorticoid receptor (GR) and/or mineralocorticoid receptor (MR) activities were suppressed. These morphological findings were confirmed in functional analysis. The cells cultured in the presence of an inhibitor of either GR or MR exhibited larger amplitudes of Ca2+ signal in response to an M channel inhibitor than did the cells in its absence, whereas the resting Ca2+ level in the former was lower than that in the latter. These results indicate that the M channel is not expressed in rat AM cells and this absence of expression may be ascribed to the suppression by glucocorticoid activity.

Serotonin Disinhibits a Caenorhabditis elegans Sensory Neuron by Suppressing Ca2+-Dependent Negative Feedback.

Neuromodulators, such as serotonin (5-HT), alter neuronal excitability and synaptic strengths, and define different behavioral states. Neuromodulator-dependent changes in neuronal activity patterns are frequently measured using calcium reporters because calcium imaging can easily be performed on intact functioning nervous systems. With only 302 neurons, the nematode Caenorhabditis elegans provides a relatively simple, yet powerful, system to understand neuromodulation at the level of individual neurons. C. elegans hermaphrodites are repelled by 1-octanol, and the initiation of these aversive responses is potentiated by 5-HT. 5-HT acts on the ASH polymodal nociceptors that sense the 1-octanol stimulus. Surprisingly, 5-HT suppresses ASH Ca2+ transients while simultaneously potentiating 1-octanol-dependent ASH depolarization. Here we further explore this seemingly inverse relationship. Our results show the following (1) 5-HT acts downstream of depolarization, through Gαq-mediated signaling and calcineurin, to inhibit L-type voltage-gated Ca2+ channels; (2) the 1-octanol-evoked Ca2+ transients in ASHs inhibit depolarization; and (3) the Ca2+-activated K+ channel, SLO-1, acts downstream of 5-HT and is a critical regulator of ASH response dynamics. These findings define a Ca2+-dependent inhibitory feedback loop that can be modulated by 5-HT to increase neuronal excitability and regulate behavior, and highlight the possibility that neuromodulator-induced changes in the amplitudes of Ca2+ transients do not necessarily predict corresponding changes in depolarization.SIGNIFICANCE STATEMENT Neuromodulators, such as 5-HT, modify behavior by regulating excitability and synaptic efficiency in neurons. Neuromodulation is often studied using Ca2+ imaging, whereby neuromodulator-dependent changes in neuronal activity levels can be detected in intact, functioning circuits. Here we show that 5-HT reduces the amplitude of depolarization-dependent Ca2+ transients in a C. elegans nociceptive neuron, through Gαq signaling and calcineurin but that Ca2+ itself inhibits depolarization, likely through Ca2+-activated K+ channels. The net effect of 5-HT, therefore, is to increase neuronal excitability through disinhibition. These results establish a novel 5-HT signal transduction pathway, and demonstrate that neuromodulators can change Ca2+ signals and depolarization amplitudes in opposite directions, simultaneously, within a single neuron.

Single cell analysis of oscillatory Ca2+ signalling in epithelial cells

Ca2+ signaling provides rich signal information on the metabolic, proliferative, and apoptotic states of cells. However, how this multiplexed signal information is encoded and decoded by cells is still unclear. Here, we report the development of a multi-scale high-content assays to reverse-engineer how Ca2+ dynamics encode cellular state information. We are using this pipeline to quantify relationships between Ca2+ oscillations dynamics and cell cycle length, growth rate, and cellular motility using both supervised and unsupervised learning methods. As a first analysis, we are using Cl.8 cells derived from Drosophila wing imaginal discs as new and useful system for investigating Ca2+ oscillatory dynamics. Strikingly, Cl.8 cells exhibit spontaneous oscillations when cultured in a chemically defined medium. This imaging pipeline enables large scale genomic investigations into which pathways, channels, and receptors modulate the oscillation frequency, duration, and amplitude of Ca2+ concentrations in cells.

RyR2 Inhibition by Dantrolene Requires both Calmodulin and FKBP12.6

Dantrolene protects cardiac muscle from arrhythmia and heart failure by inhibiting Ca2+ release from the sarcoplasmic reticulum (SR). Single-channel recordings were obtained from SR calcium release channels (RyR2) isolated from sheep and human heart and incorporated into artificial lipid bilayers. Dantrolene inhibition was measured by exposing RyR2 both in the absence and presence of exogenous 100 nM CaM. In diastolic cytoplasmic [Ca2+] (100 nM), dantrolene (50 μM) reduced the mean open probability of RyR2 to 45 ± 6% in the presence of CaM (n = 7, p<0.05, Student's paired t-test) but had no significant effect when CaM was absent (95 ± 9%, n = 20, p = 0.24). Dantrolene exhibited a hyperbolic dose response in the presence of CaM with an IC50 of 0.16 ± 0.03 μM and with a saturating relative Po (Emax) of 52 ± 4%. Emax increased to 1 as cytoplasmic Ca2+ was increased to levels >1 μM. In saponin-permeablised mouse cardiomyocytes supplemented with 100 nM CaM, dantrolene reduced the frequency and amplitude of Ca2+ waves, with an IC50 of 0.3 μM. However, when cells were depleted of CaM, dantrolene had no effect. Thus, CaM is essential for inhibitory action of dantrolene. Incubation of RyR2 to rapamycin prior to single-channel recording, known to dissociate FKBP12.6 from RyR2, removed the inhibitory action of dantrolene. This could be restored by adding FKBP12.6 to the bathing media. We conclude that the dantrolene inhibition required the presence of both CaM and FKBP12.6 in the RyR2 complex.

Ups and downs of calcium in the heart.

Contraction and relaxation of the heart result from cyclical changes of intracellular Ca2+ concentration ([Ca2+ ]i ). The entry of Ca2+ into the cell via the L-type Ca2+ current leads to the release of more from the sarcoplasmic reticulum (SR). Compared to other regulatory mechanisms such as phosphorylation, Ca2+ signalling is very rapid. However, since Ca2+ cannot be destroyed, Ca2+ signalling can only be controlled by pumping across membranes. In the steady state, on each beat, the amount of Ca2+ released from the SR must equal that taken back and influx and efflux across the sarcolemma must be equal. Any imbalance in these fluxes will result in a change of SR Ca2+ content and this provides a mechanism for regulation of SR Ca2+ content. These flux balance considerations also explain why simply potentiating Ca2+ release from the SR has no maintained effect on the amplitude of the Ca2+ transient. A low diastolic [Ca2+ ]i is essential for cardiac relaxation, but the factors that control diastolic [Ca2+ ]i are poorly understood. Recent work suggests that flux balance is also important here. In particular, decreasing SR function decreases the amplitude of the systolic Ca2+ transient and the resulting decrease of Ca2+ efflux results in an increase of diastolic [Ca2+ ]i to maintain total efflux.

Wenxin Keli diminishes Ca2+ overload induced by hypoxia/reoxygenation in cardiomyocytes through inhibiting INaL and ICaL.

An increase in the late sodium current (INaL ) causes intracellular Na+ overload and subsequently intracellular Ca2+ ([Ca2+ ]i ) overload via the stimulated reverse Na+ -Ca2+ exchange (NCX). Wenxin Keli (WXKL) is an effective antiarrhythmic Chinese herb extract, but the underlying mechanisms are unclear. The INaL , NCX current (INCX ), L-type Ca2+ current (ICaL ), and action potentials were recorded using the whole-cell patch-clamp technique in rabbit ventricular myocytes. Myocyte [Ca2+ ]i transients were measured using a dual excitation fluorescence photomultiplier system. WXKL decreased the enhanced INaL , reverse INCX , diastolic [Ca2+ ]i , and the amplitude of Ca2+ transients induced by sea anemone toxin II (ATX II, a specific INaL channel opener) in a concentration-dependent manner. Hypoxia increased INaL , INCX , and diastolic [Ca2+ ]i , and decreased amplitude of [Ca2+ ]i transients. Hypoxia-reoxygenation aggravated these changes and induced spontaneous [Ca2+ ]i transients and hypercontraction in 86% cells (6/7). The application of WXKL during hypoxia or reoxygenation periods decreased the increased INaL , INCX , and diastolic [Ca2+ ]i , and prevented those events in 82% cells (9/11) under hypoxia-reoxygenation conditions. WXKL also inhibited the ICaL in a dose-dependent manner. Furthermore, WXKL shortened the action potential duration and completely abolished ATX II-induced early afterdepolarizations from 9/9 to /9. In isolated heart electrocardiogram recordings, WXKL inhibited ischemia-reperfusion induced ventricular premature beats and tachycardia. WXKL attenuated [Ca2+ ]i overload induced by hypoxia-reoxygenation in ventricular myocytes through inhibiting INaL and ICaL and prevents arrhythmias. This could, at least partly, contribute to the antiarrhythmic effects of WXKL.

Estimating the probabilities of rare arrhythmic events in multiscale computational models of cardiac cells and tissue.

Ectopic heartbeats can trigger reentrant arrhythmias, leading to ventricular fibrillation and sudden cardiac death. Such events have been attributed to perturbed Ca2+ handling in cardiac myocytes leading to spontaneous Ca2+ release and delayed afterdepolarizations (DADs). However, the ways in which perturbation of specific molecular mechanisms alters the probability of ectopic beats is not understood. We present a multiscale model of cardiac tissue incorporating a biophysically detailed three-dimensional model of the ventricular myocyte. This model reproduces realistic Ca2+ waves and DADs driven by stochastic Ca2+ release channel (RyR) gating and is used to study mechanisms of DAD variability. In agreement with previous experimental and modeling studies, key factors influencing the distribution of DAD amplitude and timing include cytosolic and sarcoplasmic reticulum Ca2+ concentrations, inwardly rectifying potassium current (IK1) density, and gap junction conductance. The cardiac tissue model is used to investigate how random RyR gating gives rise to probabilistic triggered activity in a one-dimensional myocyte tissue model. A novel spatial-average filtering method for estimating the probability of extreme (i.e. rare, high-amplitude) stochastic events from a limited set of spontaneous Ca2+ release profiles is presented. These events occur when randomly organized clusters of cells exhibit synchronized, high amplitude Ca2+ release flux. It is shown how reduced IK1 density and gap junction coupling, as observed in heart failure, increase the probability of extreme DADs by multiple orders of magnitude. This method enables prediction of arrhythmia likelihood and its modulation by alterations of other cellular mechanisms.

The calcium transient characteristics induced by fluid shear stress affect the osteoblast proliferation

Ca2+ signaling is essential for bone metabolism. Fluid shear stress (FSS), which can induce a rapid release of calcium from endoplasmic reticulum (ER) to produce calcium transients, plays a significant role in osteoblast proliferation and differentiation. However, it is still unclear of how calcium transients induced by FSS activating a number of downstream signals which subsequently regulate cell functions. In this study, we performed a group of Ca2+ transients models, which were induced by FSS to investigate the effects of different magnitudes of Ca2+ transients in osteoblast proliferation. Further, we performed a global proteomic profile of MC3T3-E1 cells in different Ca2+ transients models stimulated by FSS. GO enrichment and KEGG pathway analysis revealed that the TCA cycle was activated in the proliferating process. The activation of TCA needed mitochondrial Ca2+ uptake which were influenced by the amplitude of Ca2+ transients induced by FSS. Our work elucidate that osteoblast proliferation induced by FSS was related to the magnitude of calcium transients, which further activated energetic metabolism signaling pathway. This work revealed further understanding the mechanism of osteoblast proliferation induced by mechanic loading and help us to design new methods for osteoporosis therapy.

P2X receptor overexpression induced by soluble oligomers of amyloid beta peptide potentiates synaptic failure and neuronal dyshomeostasis in cellular models of Alzheimer's disease

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aβ). Because Aβ increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aβ and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aβ oligomers (0.5 μM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aβ. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aβ. Taken together, our findings suggest that Aβ disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.