Nuclear Ca2 Research Articles

Circulation Research “In This Issue” Anthology

<i>Circulation Research</i> “In This Issue” Anthology

Measuring intranuclear and nuclear envelope [Ca(2+)] vs. cytosolic [Ca (2+)

Nuclear Ca(2+) regulates key cellular processes, including gene expression, apoptosis, assembly of the nuclear envelope, and nucleocytoplasmic transport. Quantification of subcellularly resolved Ca(2+) signals is, therefore, essential for understanding physiological and pathological processes in various cell types. However, the properties of commonly used Ca(2+)-fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca(2+)-dependent fluorescence changes into quantitative changes of Ca(2+) concentration. Here, we describe technical approaches for reliable subcellular quantification of [Ca(2+)] in the cytoplasm vs. the nucleus and the nuclear envelope by in situ calibration of fluorescein-derived fluorescent indicators Fluo-4 and Fluo-5N.

Design and application of light-activated probes for cellular signaling.

Multiple reports have described the presence of functional G protein-coupled receptors (GPCRs) in the perinuclear/nuclear membranes of many cell types where they are able to modulate nuclear Ca(2+) influx, transcription initiation, and gene expression. Because GPCRs represent "some of the most promising targets for drug development" a better understanding of their roles, not only at the cell membrane but also at the nuclear level, in healthy and disease states, will certainly generate new avenues for therapeutic intervention. The photo-triggered release of biologically active compounds has been regarded as one of the most effective methods for inducing an in vitro-controlled biochemical or physiological response. Here, we describe various methodologies and alternatives related to the conception of inert biologically active peptides through the incorporation of photo-triggered groups at key positions of the native peptide sequence.

Measuring spatial and temporal Ca2+ signals in Arabidopsis plants.

Developmental and environmental cues induce Ca(2+) fluctuations in plant cells. Stimulus-specific spatial-temporal Ca(2+) patterns are sensed by cellular Ca(2+) binding proteins that initiate Ca(2+) signaling cascades. However, we still know little about how stimulus specific Ca(2+) signals are generated. The specificity of a Ca(2+) signal may be attributed to the sophisticated regulation of the activities of Ca(2+) channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca(2+) signals at both the tissue and cellular levels. Genetically encoded Ca(2+) indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca(2+) signals. Here we describe instructions for the use of two Ca(2+) detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca(2+) imaging and case12 based live cell confocal fluorescence Ca(2+) imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca(2+) signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca(2+) signals at a high resolution.

Living a healthier lifestyle.

The roles of diet, activity, and exercise have long been recognized as essential to a healthy lifestyle. As Plato stated, “Lack of activity destroys the good condition of every human being, while movement and methodical physical exercise save it and preserve it.” In his Physiology is Medicine

Decoding calcium signaling across the nucleus.

Calcium (Ca(2+)) is an important multifaceted second messenger that regulates a wide range of cellular events. A Ca(2+)-signaling toolkit has been shown to exist in the nucleus and to be capable of generating and modulating nucleoplasmic Ca(2+) transients. Within the nucleus, Ca(2+) controls cellular events that are different from those modulated by cytosolic Ca(2+). This review focuses on nuclear Ca(2+) signals and their role in regulating physiological and pathological processes.

Calcineurin mediates synaptic scaling via synaptic trafficking of Ca2+-permeable AMPA receptors.

Author SummarySynaptic scaling is a form of homeostatic plasticity that normalizes the strength of synapses (the structure that allows nerve cells to communicate) and is triggered by chronic inhibition of neuronal activity. Although extensive studies have been conducted, the molecular mechanism of this synaptic adaptation is not understood. Using cultured cortical neurons, we show that chronic inhibition of neuronal activity reduces calcium influx into neurons, which, in turn, decreases the activity of the calcium-dependent phosphatase calcineurin. These changes lead to an increase in GluA1-containing, calcium-permeable AMPA receptors, which mediate communication at the synapse. Newly inserted calcium-permeable AMPA receptors restore calcium currents, which enhance synaptic strength and recover calcium signaling. We also show that inhibition or activation of calcineurin activity is sufficient to induce or block synaptic scaling, respectively, suggesting that calcineurin is an important mediator of homeostatic synaptic plasticity. Taken together, our findings show that synaptic scaling is a homeostatic process that not only enhances synaptic transmission but also maintains calcium signaling in neurons under activity deprivation.

Localization and projected role of phosphatidylinositol 4-kinases IIα and IIβ in inositol 1,4,5-trisphosphate-sensitive nucleoplasmic Ca2+ store vesicles

Phosphatidylinositol (PI) kinases are key molecules that participate in the phosphoinositide signaling in the cytoplasm. Despite the accumulating evidence that supports the existence and operation of independent PI signaling system in the nucleus, the exact location of the PI kinases inside the nucleus is not well defined. Here we show that PI4-kinases IIα and IIβ, which play central roles in PI(4,5)P2 synthesis and PI signaling, are localized in numerous small nucleoplasmic vesicles that function as inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ stores. This is in accord with the past results that showed the localization of PI4(P)5-kinases that are essential in PI(4,5)P2 production and PI(4,5)P2 in nuclear matrix. Along with PI(4,5)P2 that also exists on the nucleoplasmic vesicle membranes, the localization of PI4-kinases IIα and IIβ in the nucleoplasmic vesicles strongly implicates the vesicles to the PI signaling as well as the Ins(1,4,5)P3-depenent Ca2+ signaling in the nucleus. Accordingly, the nucleoplasmic vesicles indeed release Ca2+ rapidly in response to Ins(1,4,5)P3. Further, the Ins(1,4,5)P3-induced Ca2+ release studies suggest that PI4KIIα and IIβ are localized near the Ins(1,4,5)P3 receptor (Ins(1,4,5)P3R)/Ca2+ channels on the Ca2+ store vesicle membranes. In view of the widespread presence of the Ins(1,4,5)P3-dependent Ca2+ store vesicles and the need to fine-control the nuclear Ca2+ concentrations at multiple sites along the chromatin fibers in the nucleus, the existence of the key PI enzymes in the Ins(1,4,5)P3-dependent nucleoplasmic Ca2+ store vesicles appears to be in perfect harmony with the physiological roles of the PI kinases in the nucleus.

Nuclear BK channels regulate gene expression via the control of nuclear calcium signaling.

Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal ion channels in the regulation of Ca2+ is well established. In contrast, surprisingly less is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally blockade of nuclear BK channels (nBK channels) induces NE-derived Ca2+ release, nucleoplasmic Ca2+ elevation, and cAMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+-sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+-dependent manner. These results suggest that nBK channel functions as a molecular linker between neuronal activity and nuclear Ca2+ to convey the signals from synapse to nucleus and is a new modulator for synaptic activity-dependent neuronal functions at the NE level.

Properties of Inositol 1,4,5-Trisphosphate Receptors in the Nuclei of CNS Neurons in Rats

We studied the properties of inositol 1,4,5-trisphosphate receptors (InsP3Rs) in the membranes of the nuclear envelope of CNS neurons in rats. In cells with a maximum level of expression of InsP3Rs, (hippocampal and cerebellar neurons), these receptors are localized predominantly in the inner nuclear membrane. This allows us to hypothesize that the nuclear Ca2+ store is involved in regulation of expression of Ca2+-dependent genes. Applications of agonists of InsP3Rs (InsP3 and Ca2+ in concentrations below 1 μM) evoked rapid activation of channels of InsP3Rs followed by their steady-state activity. Application of [Ca2+] ≥ 1 μM did not influence significantly the characteristics of peak responses, but then the InsP3Rs lose sensitivity almost completely during several seconds. Therefore, the Ca2+ dependence of steady-state activity of these neurons was bell-shaped. Within physiological limits of concentrations of intracellular Ca2+, the peak activity of the above-mentioned receptors was not inhibited by applications of agonists. These findings explain the main contradiction between the data obtained recently by different research groups. At the same time, the basic nature of kinetic properties of InsP3Rs under conditions of various experimental models needs further investigation.

Ion channels of the nuclear membrane of hippocampal neurons

Rise in Ca(2+) concentration in the nucleus affects gene transcription and has been implicated in neuroprotection, transcription-dependent neuronal plasticity, and pain modulation, but the mechanism of regulation of nuclear Ca(2+) remains poorly understood. The nuclear envelope is a part of the endoplasmic reticulum and may be one of the sources of nuclear Ca(2+) . Here, we studied ion channels in the nuclear membrane of hippocampal neurons using the patch-clamp technique. We have found that the nuclear membrane of CA1 pyramidal and dentate gyrus granule (DG), but not CA3 pyramidal neurons, was enriched in functional inositol 1,4,5-trisphosphate receptors/Ca(2+) -release channels (IP3 Rs) localized mainly in the inner nuclear membrane. A single nuclear ryanodine receptor (RyR) has been detected only in DG granule neurons. Nuclei of the hippocampal neurons also expressed a variety of spontaneously active cation and anion channels specific for each type of neuron. In particular, large-conductance ion channels selective for monovalent cations (LCC) were coexpressed with IP3 Rs. These data suggest that: (1) the nuclear membranes of hippocampal neurons contain distinct sets of ion channels, which are specific for each type of neuron; (2) IP3 Rs, but not RyRs are targeted to the inner nuclear membrane of CA1 pyramidal and DG granule, but they were not found in the nuclear membranes of CA3 pyramidal neurons; (3) the nuclear envelope of these neurons is specialized to release Ca(2+) into the nucleoplasm which may amplify Ca(2+) signals entering the nucleus from the cytoplasm or generate Ca(2+) transients on its own; (4) LCC channels are an integral part the of Ca(2+) -releasing machinery providing a route for counterflow of К(+) and thereby facilitating Ca(2+) movement in and out of the Ca(2+) store.

Effect of hypoxia on nuclear high affinity Ca2+ ‐ATPase activity and nuclear Ca2+ ‐influx in the cerebral cortex of newborn piglets (888.2)

Nuclear calcium signals control a number of critical nuclear functions including regulation of transcription factors, cell cycle regulation, gene transcription and DNA replication. Previous studies have shown that during hypoxia, there is increase in intracellular Ca2+‐and in nuclear high affinity Ca2+‐ATPase activity in the cerebral cortex of newborn piglets.We test the hypothesis that hypoxia results in increased activity of high affinity Ca2+‐ATPase and increased concentration of Ca2+‐influx in cortical neuronal nuclei of newborn piglets, and this hypoxia‐induced increase is Src kinase mediated.Piglets were divided into normoxic (Nx, n=4), hypoxic (Hx, n=4), and hypoxic pretreated with Src kinase inhibitor (Hx+Srci, n=4). Hypoxia was induced by decreasing FiO2 to 0.07 for 1 hr. Src kinase inhibitor (PP2, 1.0 mg/kg i.v.) was administered 30 min prior to hypoxia. Hypoxia was documented by levels of ATP and phosphocreatine (PCr). Nuclear 45Ca2+ concentration and activity of high affinity Ca2+‐ATPase was determined at 37°C for 30 min.Nuclear 45Ca2+ ‐influx (pmoles/mg protein) was 3.95±0.57 in Nx , 13.36±1.56 in Hx (p&lt;0.05) and 9.61±2.76 in the Hx+Srci group. Ca2+‐ATPase activity increased following hypoxia: from 244.66±73.55 nmol Pi/mg protein/h in the normoxic group to 426.33±94.99 nmol Pi/mg protein/h in the hypoxic group (p&lt;0.05 vs. normoxic group). In the Hx+Srci group, the high affinity Ca2+‐ATPase activity increased to 354.25±62.88 nmol Pi/mg protein/h (p&lt;0.05 vs. hypoxic group). The data show that administration of PP2, a selective inhibitor of Src kinase, prevents the hypoxia‐induced increase in nuclear 45Ca2+ ‐influx concentration and Ca2+‐ATPase activity.We conclude that the hypoxia‐induced increase in nuclear membrane high affinity Ca2+‐ATPase activity is Src kinase‐mediated activation of the high affinity Ca2+‐ATPase, a mechanism of ATP‐dependent Ca2+uptake may lead to increase in intranuclear Ca2+during hypoxia.Grant Funding Source: NIH HD200337

Calcium and IP3 dynamics in cardiac myocytes: experimental and computational perspectives and approaches

Calcium plays a crucial role in excitation-contraction coupling (ECC), but it is also a pivotal second messenger activating Ca2+-dependent transcription factors in a process termed excitation-transcription coupling (ETC). Evidence accumulated over the past decade indicates a pivotal role of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release in the regulation of cytosolic and nuclear Ca2+ signals. IP3 is generated by stimulation of plasma membrane receptors that couple to phospholipase C (PLC), liberating IP3 from phosphatidylinositol 4,5-bisphosphate (PIP2). An intriguing aspect of IP3 signaling is the presence of the entire PIP2-PLC-IP3 signaling cascade as well as the presence of IP3Rs at the inner and outer membranes of the nuclear envelope (NE) which functions as a Ca2+ store. The observation that the nucleus is surrounded by its own putative Ca2+ store raises the possibility that nuclear IP3-dependent Ca2+ release plays a critical role in ETC. This provides a potential mechanism of regulation that acts locally and autonomously from the global cytosolic Ca2+ signal underlying ECC. Moreover, there is evidence that: (i) the sarcoplasmic reticulum (SR) and NE are a single contiguous Ca2+ store; (ii) the nuclear pore complex is the major gateway for Ca2+ and macromolecules to pass between the cytosol and the nucleoplasm; (iii) the inner membrane of the NE hosts key Ca2+ handling proteins including the Na+/Ca2+ exchanger (NCX)/GM1 complex, ryanodine receptors (RyRs), nicotinic acid adenine dinucleotide phosphate receptors (NAADPRs), Na+/K+ ATPase, and Na+/H+ exchanger. Thus, it appears that the nucleus represents a Ca2+ signaling domain equipped with its own ion channels and transporters that allow for complex local Ca2+ signals. Many experimental and modeling approaches have been used for the study of intracellular Ca2+ signaling but the key to the understanding of the dual role of Ca2+ mediating ECC and ECT lays in quantitative differences of local [Ca2+] in the nuclear and cytosolic compartment. In this review, we discuss the state of knowledge regarding the origin and the physiological implications of nuclear Ca2+ transients in different cardiac cell types (adult atrial and ventricular myocytes) as well as experimental and mathematical approaches to study Ca2+ and IP3 signaling in the cytosol and nucleus. In particular, we focus on the concept that highly localized Ca2+ signals are required to translocate and activate Ca2+-dependent transcription factors (e.g., nuclear factor of activated T-cells, NFAT; histone deacetylase, HDAC) through phosphorylation/dephosphorylation processes.

DREAM Controls the On/Off Switch of Specific Activity-Dependent Transcription Pathways

Changes in nuclear Ca(2+) homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K(+) channel interacting protein 3), is a Ca(2+)-binding protein that binds DNA and represses transcription in a Ca(2+)-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca(2+)-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory.

Cell Proliferation and Migration Induced by Angiotensin-II is Mediated by ACE

We have demonstrated that the Angiotensin I Converting Enzyme (ACE) acts as a membrane receptor for Angiotensin II (AngII). Upon binding to ACE with high affinity, AngII activates a signaling cascade that generates inositol 1,4,5-triphosphate (InsP3), culminating in intracellular Ca2+ increase. The goal of this work was to investigate cellular function that AngII plays through ACE activation. For that, we used CHO-ACE, CHO-AT1 and melanoma cell line (Tm-5), as well as, confocal microscopy, immunofluorescence, western blotting, small interfering RNA (siRNA) and BrDU assay. We found that AngII binds to ACE and this complex is internalized through clathrin, to trigger preferential nucleoplasmic Ca2+ increase. Since nuclear Ca2+ is known to regulate cell growth, we then investigated whether the proliferative response induced by AngII is mediated by ACE activation. We verified that silencing either ACE or clathrin reduced cell proliferation stimulated by AngII by 33% or 71% respectively, compared to control (p<0.001). In melanoma cell line (Tm-5), that was known to expresses mainly ACE instead of the classical AngII receptor, the AT1, AngII increased cell migration by 75.7 ± 2% compared to control condition (p<0.05). This data correlated with reduced focal adhesion formation induced by AngII in the same cell line (154 ± 23.9 a.u. for control vs, 54.9 ± 9.8 a.u. for AngII, p<0.05), Together, these results indicate a new role for ACE in cell proliferation and migration induced by AngII. Moreover, these findings point ACE signaling as a novel therapeutic target for cancer treatment.

Alterations of Nuclear Ca2+-Dependent Signalling in Heart Failure

A hallmark of heart failure is impaired Ca2+ handling of cardiomyocytes. We previously showed that specific alterations in nuclear Ca2+ handling precede changes in cytoplasmic Ca2+ handling as heart failure progresses. However, a direct link between changes of nucleoplasmic Ca2+ handling and altered excitation-transcription coupling during the heart failure progression was not previously established. We thus characterized changes of nuclear Ca2+ handling and the activation of nuclear Ca2+-dependent transcription factors under low and high pacing frequencies at the early and late stage of hypertrophy in mouse model of pressure overload.Ventricular cardiomyocytes were isolated 1 and 7 weeks after transverse aortic constriction (TAC) in adult wild-type mice. Subcellular [Ca2+] transients were recorded in electrically stimulated CMs loaded with Fluo-4/AM. Phosphorylation levels of CaMKII and nuclear accumulation of HDAC4 were quantified by immunocytostaining.During the early remodelling (i.e. 1 week after TAC intervention) - in contrast to diastolic [Ca2+] in the cytoplasm - diastolic [Ca2+] in the nucleus was already elevated at very low stimulation rate (0.5 Hz) as compared to the non-failing group, and than overproportionally increased with faster stimulation rates. In failing cardiomyocytes (7 weeks after TAC intervention), the changes in nucleoplasmic and cytoplasmic diastolic [Ca2+] were qualitatively comparable, though the increase was more pronounced in the nuclear compartment. High pacing frequency caused significantly higher phosphorylation of CaMKII and corresponding HDAC4 translocation in cardiomyocytes from hypertrophic hearts compared to healthy controls, with the significantly higher increase in CaMKII phosphorylation in the nucleoplasmic compartment as compared to cytoplasm.In conclusion, we found that the increased stimulation frequency led to a higher build-up of diastolic [Ca2+] in cardiomyocytes from hypertrophied hearts, especially in the nucleoplasmic compartment, which may be involved in the dysregulation of Ca2+-dependent gene transcription and progression of adverse cardiac remodeling.

Abstract 18006: Nuclear Calcineurin is a Sensor for Detecting Ca2+ Release From the Nuclear Envelope via InsP3R

Background: Recent evidence demonstrates that not only NFAT, but also calcineurin (CN) is translocated to the nucleus upon hypertrophic stimulation. It was also shown that calpain-mediated degradation caused a constitutive active CN. We hypothesised 1) that nuclear CN is an intranuclear Ca2+ sensor, 2) that the nucleus in cardiac myocytes is a Ca2+ microdomain largely independent of cytosolic Ca2+ alterations and that 3) inhibition of nuclear translocation of CN is a therapeutic strategy to prevent hypertrophy. Methods: Employing a transgene mouse model with conditional calpastatin overexpression (resulting in calpain inhibition), different adenoviral CN mutants and confocal microscopy in isolated adult cardiac myocytes we investigated CN translocation and nuclear Ca2+ transients. Assessment of cardiac function in transgenic animals was performed by MRI. Results: We could demonstrate that chronic Ang II stimulation of mice caused calpain-mediated degradation of CN resulting in a constitutive active CN with nuclear translocation. The constitutive active CN in the nucleus escaped degradation by the UPS and sustained an ongoing hypertrophic response, even after removal of Ang II. Transgenic inhibition of calpain by calpastatin overexpression prevented proteolysis of CN and allowed for relocation of CN from the nucleus back to the cytosol and regression of hypertrophy after removal of Ang II. Inhibition of the interaction between CN and the transport protein importin ß1 by an inhibitory peptide prevented nuclear translocation of activated CN. The synthetic peptide prevented myocardial hypertrophy in vivo. We were able to demonstrate that Ang II increases nuclear Ca2+ transients via nuclear InsP3 receptors. We also showed that Ca2+ levels are constantly elevated during diastole (up to 0.2 μM) in Ang II stimulated cardiac myocytes and that CN is able to act as nuclear Ca2+ sensor detecting this local Ca2+ release from the nuclear envelope via InsP3R. Nuclear CN mutants that are defective for Ca2+ activation failed to activate NFAT dependent transcription. Conclusion: These results provide an explanation how Ca2+ and CN can regulate transcription in cardiac myocytes in response to neurohumoral signals apart from Ca2+ changes in contraction regulation.

Early alterations of nuclear Ca2+-dependent signalling in cardiac hypertrophy

Early alterations of nuclear Ca2+-dependent signalling in cardiac hypertrophy

Imaging of Mitochondrial and Nuclear Ca2+ Dynamics in Arabidopsis Roots

Here we report a method for analyzing mitochondrial Ca(2+) dynamics in plant root cells by means of the newly generated cameleon probe 4mt-YC3.6. The use of plants expressing both nuclear- and mitochondrial-targeted cameleon, along with the resolution of confocal laser scanning microscopy (CLSM), allow simultaneous recordings of mitochondrial and nuclear Ca(2+) dynamics within the same cell.

Live Cell Imaging of Cytoplasmic and Nuclear Ca2+ Dynamics in Arabidopsis Roots

Approaches for Ca(2+) measurement require data recording with high spatiotemporal resolution. This is achieved by combined usage of locally targeted genetically encoded calcium indicators (GECIs) and confocal laser scanning microscopy (CLSM). This protocol provides instructions for setting up a CLSM-based experiment to measure cytosolic and nuclear Ca(2+) dynamics in Arabidopsis lines expressing Yellow Cameleon Ca(2+) indicators.