Nucleoplasmic Ca2 Research Articles

Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure.

Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) versus perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF versus PN mitochondria in pressure overload-induced cardiac remodelling in mice, and in non-failing and failing human cardiomyocytes. We demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to depolarization of mitochondrial membrane potential, reactive oxygen species generation and impairment in Ca2+ uptake compared with IF mitochondria at baseline and under physiological stress protocol. We also demonstrate, for the first time to our knowledge, that under normal conditions PN mitochondrial Ca2+ uptake shapes nucleoplasmic Ca2+ transients (CaTs) and limits nucleoplasmic Ca2+ loading. The loss of PN mitochondrial Ca2+ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signalling and alleviation of altered excitation–transcription, which could be an important therapeutic approach to prevent adverse cardiac remodelling.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus.

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.

Loss of autophagy protein ATG5 impairs cardiac capacity in mice and humans through diminishing mitochondrial abundance and disrupting Ca2+ cycling.

AimsAutophagy protects against the development of cardiac hypertrophy and failure. While aberrant Ca2+ handling promotes myocardial remodelling and contributes to contractile dysfunction, the role of autophagy in maintaining Ca2+ homeostasis remains elusive. Here, we examined whether Atg5 deficiency-mediated autophagy promotes early changes in subcellular Ca2+ handling in ventricular cardiomyocytes, and whether those alterations associate with compromised cardiac reserve capacity, which commonly precedes the onset of heart failure.Methods and resultsRT–qPCR and immunoblotting demonstrated reduced Atg5 gene and protein expression and decreased abundancy of autophagy markers in hypertrophied and failing human hearts. The function of ATG5 was examined using cardiomyocyte-specific Atg5-knockout mice (Atg5−/−). Before manifesting cardiac dysfunction, Atg5−/− mice showed compromised cardiac reserve in response to β-adrenergic stimulation. Consequently, effort intolerance and maximal oxygen consumption were reduced during treadmill-based exercise tolerance testing. Mechanistically, cellular imaging revealed that Atg5 deprivation did not alter spatial and functional organization of intracellular Ca2+ stores or affect Ca2+ cycling in response to slow pacing or upon acute isoprenaline administration. However, high-frequency stimulation exposed stunted amplitude of Ca2+ transients, augmented nucleoplasmic Ca2+ load, and increased CaMKII activity, especially in the nuclear region of hypertrophied Atg5−/− cardiomyocytes. These changes in Ca2+ cycling were recapitulated in hypertrophied human cardiomyocytes. Finally, ultrastructural analysis revealed accumulation of mitochondria with reduced volume and size distribution, meanwhile functional measurements showed impaired redox balance in Atg5−/− cardiomyocytes, implying energetic unsustainability due to overcompensation of single mitochondria, particularly under increased workload.ConclusionLoss of cardiac Atg5-dependent autophagy reduces mitochondrial abundance and causes subtle alterations in subcellular Ca2+ cycling upon increased workload in mice. Autophagy-related impairment of Ca2+ handling is progressively worsened by β-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve.

Inositol Trisphosphate Receptors and Nuclear Calcium in Atrial Fibrillation.

The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca2+ regulates gene expression, but the nature and significance of nuclear Ca2+-changes in AF are largely unknown. To elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca2+ ([Ca2+]Nuc) and CaMKII (Ca2+/calmodulin-dependent protein kinase-II)-related signaling. Atrial cardiomyocytes were isolated from control and AF dogs (kept in AF by atrial tachypacing [600 bpm × 1 week]). [Ca2+]Nuc and cytosolic [Ca2+] ([Ca2+]Cyto) were recorded via confocal microscopy. Diastolic [Ca2+]Nuc was greater than [Ca2+]Cyto under control conditions, while resting [Ca2+]Nuc was similar to [Ca2+]Cyto; both diastolic and resting [Ca2+]Nuc increased with AF. IP3R (Inositol-trisphosphate receptor) stimulation produced larger [Ca2+]Nuc increases in AF versus control cardiomyocytes, and IP3R-blockade suppressed the AF-related [Ca2+]Nuc differences. AF upregulated nuclear protein expression of IP3R1 (IP3R-type 1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot) while decreasing the nuclear/cytosolic expression ratio for HDAC4 (histone deacetylase type-4). Isolated atrial cardiomyocytes tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca2+]Nuc changes and L-type calcium current decreases versus 1-Hz-paced cardiomyocytes; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IP3R1. Nuclear/cytosolic HDAC4 expression ratio was decreased by 3-Hz pacing, while nuclear CaMKII phosphorylation was increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP3R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial cardiomyocytes from AF patients, nuclear IP3R1-expression was significantly increased, with decreased nuclear/nonnuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by luciferase assay) and was downregulated in AF atrial cardiomyocytes; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca2+-loading. AF increases atrial-cardiomyocyte nucleoplasmic [Ca2+] by IP3R1-upregulation involving miR-26a, leading to enhanced IP3R1-CaMKII-HDAC4 signaling and L-type calcium current downregulation. Graphic Abstract: A graphic abstract is available for this article.

Crosstalk between FGF23- and angiotensin II-mediated Ca2+ signaling in pathological cardiac hypertrophy.

Heart failure (HF) manifestation and progression are driven by systemic activation of neuroendocrine signaling cascades, such as the renin-angiotensin aldosterone system (RAAS). Fibroblast growth factor 23 (FGF23), an endocrine hormone, is linked to HF and cardiovascular mortality. It is also a mediator of left-ventricular hypertrophy(LVH). In vivo, high circulating levels of FGF23 are associated with an altered systemic RAAS response. FGF23 is proposed to trigger pathological signaling mediated by Ca2+-regulated transcriptional pathways. In the present study, we investigated Ca2+-dependent signaling of FGF23 in ventricular cardiomyocytes and its association with angiotensin II (ATII). In neonatal rat ventricular myocytes (NRVMs), both ATII and FGF23 induced hypertrophy as observed by an increase in cell area and hypertrophic gene expression. Furthermore, FGF23 activates nuclear Ca2+-regulated CaMKII-HDAC4 pathway, similar to ATII. In addition to a global increase in cytoplasmic Ca2+, FGF23, like ATII, induced inositol 1, 4, 5-triphosphate (IP3)-induced Ca2+ release from the nucleoplasmic Ca2+ store, associated with cellular hypertrophy. Interestingly, ATII receptor antagonist, losartan, significantly attenuated FGF23-induced changes in Ca2+ homeostasis and cellular hypertrophy suggesting an involvement of ATII receptor-mediated signaling. In addition, application of FGF23 increased intracellular expression of ATII peptide and its secretion in NRVMs, confirming the participation of ATII. In conclusion, FGF23 and ATII share a common mechanism of IP3-nuclear Ca2+-dependent cardiomyocyte hypertrophy. FGF23-mediated cellular hypertrophy is associated with increased production and secretion of ATII by cardiomyocytes. These findings indicate a pathophysiological role of the cellular angiotensin system in FGF23-induced hypertrophy in ventricular cardiomyocytes.

Na+/Ca2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca2+ regulation during neuronal differentiation

Nuclear envelope (NE) is a Ca2+-storing organelle controlling neuronal differentiation through nuclear Ca2+ concentrations ([Ca2+]n). However, how [Ca2+]n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na+/Ca2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3’K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation. nuNCX1 activation by Na+-free perfusion induced a time-dependent activation of nuclear-resident PI3K/Akt pathway in isolated nuclei. To discriminate the contribution of nuNCX1 from those of plasma membrane NCX, we generated a chimeric protein composed of the fluorophore EYFP, the exchanger inhibitory peptide, and the nuclear localization signal, named XIP-NLS. Fura-2 measurements on single nuclei and patch-clamp experiments in whole-cell configuration showed that XIP-NLS selectively inhibited nuNCX1. Once it reached the nuclear compartment, XIP-NLS increased the nucleoplasmic Ca2+ peak elicited by ATP and reduced Akt phosphorylation, GAP-43 and MAP-2 expression through nuclear-resident PTEN induction. Furthermore, in accordance with the prevention of the neuronal phenotype, XIP-NLS significantly reduced TTX-sensitive Na+ currents and membrane potential during neuronal differentiation. The selective inhibition of nuNCX1 by XIP-NLS increased the percentage of β III tubulin-positive immature neurons in mature cultures of MAP-2-positive cortical neurons, thus unraveling a new function for nuNCX1 in regulating neuronal differentiation through [Ca2+]n-dependent PTEN/PI3K/Akt pathway.

Three-dimensional imaging reveals endo(sarco)plasmic reticulum-containing invaginations within the nucleoplasm of muscle.

The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca2+ homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine C2C12 muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca2+ regulatory channels, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca2+ release-activated Ca2+ channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca2+ regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.

Intracellular Angiotensin-II Interacts With Nuclear Angiotensin Receptors in Cardiac Fibroblasts and Regulates RNA Synthesis, Cell Proliferation, and Collagen Secretion.

BackgroundCardiac fibroblasts play important functional and pathophysiological roles. Intracellular (“intracrine”) angiotensin‐II (Ang‐II) signaling regulates intercellular communication, excitability, and gene expression in cardiomyocytes; however, the existence and role of intracrine Ang‐II signaling in cardiac fibroblasts is unstudied. Here, we evaluated the localization of Ang‐II receptors on atrial fibroblast nuclei and associated intracrine effects of potential functional significance.Methods and ResultsImmunoblots of subcellular protein‐fractions from isolated canine atrial fibroblasts indicated the presence of nuclear Ang‐II type 1 receptors (AT1Rs) and Ang‐II type 2 receptors (AT2Rs). Fluorescein isothiocyanate–Ang‐II binding displaceable by AT1R‐ and AT2R‐blockers was present on isolated fibroblast nuclei. G‐protein subunits, including Gαq/11, Gαi/3, and Gβ, were observed in purified fibroblast nuclear fractions by immunoblotting and intact‐fibroblast nuclei by confocal immunocytofluorescence microscopy. Nuclear AT1Rs and AT2Rs regulated de novo RNA synthesis ([α32P]UTP incorporation) via IP3R‐ and NO‐dependent pathways, respectively. In intact cultured fibroblasts, intracellular Ang‐II release by photolysis of a membrane‐permeable caged Ang‐II analog led to IP3R‐dependent nucleoplasmic Ca2+‐liberation, with IP3R3 being the predominant nuclear isoform. Intracellular Ang‐II regulated fibroblast proliferation ([3H]thymidine incorporation), collagen‐1A1 mRNA‐expression, and collagen secretion. Intracellular Ang‐II and nuclear AT1R protein levels were significantly increased in a heart failure model in which atrial fibrosis underlies atrial fibrillation.ConclusionsFibroblast nuclei possess AT1R and AT2R binding sites that are coupled to intranuclear Ca2+‐mobilization and NO liberation, respectively. Intracellular Ang‐II signaling regulates fibroblast proliferation, collagen gene expression, and collagen secretion. Heart failure upregulates Ang‐II intracrine signaling‐components in atrial fibroblasts. These results show for the first time that nuclear angiotensin‐II receptor activation and intracrine Ang‐II signaling control fibroblast function and may have pathophysiological significance.

Sequences within the C Terminus of the Metabotropic Glutamate Receptor 5 (mGluR5) Are Responsible for Inner Nuclear Membrane Localization

Traditionally, G-protein-coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However, recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5 (mGluR5) is concentrated at the inner nuclear membrane (INM) where it mediates Ca2+ changes in the nucleoplasm by coupling with Gq/11 Here, we identified a region within the C-terminal domain (amino acids 852-876) that is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known nuclear localization signal motifs, they represent a new motif for INM trafficking. mGluR5 is also trafficked to the plasma membrane where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with chromatin. Thus, mGluR5 is perfectly positioned to regulate nucleoplasmic Ca2+in situ.

Enhanced nucleoplasmic Ca2 + signaling in ventricular myocytes from young hypertensive rats

Arterial hypertension causes left ventricular (LV) myocyte hypertrophy. Alterations in nuclear Ca2+ may be involved in regulation of histone acetylation, transcription and hypertrophy. Regulation of nuclear Ca2+ in hypertension, however, is unknown. Therefore, we elucidated cellular mechanisms underlying nuclear Ca2+ regulation in LV myocytes from hypertensive versus normotensive rats and evaluated possible consequences for Ca2+-dependent regulation of histone acetylation. LV myocytes and myocyte nuclei were isolated from young spontaneously hypertensive rats (SHR) shortly after development of hypertension. Normotensive Wistar-Kyoto rats (WKY) served as controls. Cytoplasmic and nucleoplasmic Ca2+ transients (CaTs) were imaged simultaneously using linescan confocal microscopy and Fluo-4. LV myocytes and nuclei from SHR exhibited hypertrophy. Cytoplasmic and nucleoplasmic CaTs were increased in SHR. The increase in nucleoplasmic Ca2+, however, exceeded the increase in cytoplasmic Ca2+, indicating enhanced nuclear Ca2+ signaling in SHR. Ca2+ load of sarcoplasmic reticulum and perinuclear Ca2+ stores was also increased in SHR, while fractional release from both stores remained unchanged. Intranuclear Ca2+ propagation was accelerated in SHR, associated with preserved density of nuclear envelope invaginations and elevated nuclear expression of nucleoporins and SR-Ca2+-ATPase, SERCA2a. Nuclear Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) expression was elevated and histone deacetylases exhibited redistribution from nucleus to cytosol associated with increased histone acetylation in SHR. Thus, in early hypertension, there is remodeling of nuclear Ca2+ handling resulting in enhanced nuclear Ca2+ signaling. Enhanced nuclear Ca2+ signaling, in turn, increases nuclear localization and activity of CaMKIIδ driving nuclear export of histone deacetylases and increased histone acetylation.

A stromal interaction molecule 1 variant up-regulates matrix metalloproteinase-2 expression by strengthening nucleoplasmic Ca2+ signaling

Very recent studies hold promise to reveal the role of stromal interaction molecule 1 (STIM1) in non-store-operated Ca2+ entry. Here we showed that in contrast to cytoplasmic membrane redistribution as previously noted, human umbilical vein endothelial STIM1 with a T-to-C nucleotide transition resulting in an amino acid substitution of leucine by proline in the signal peptide sequence translocated to perinuclear membrane upon intracellular Ca2+ depletion, amplified nucleoplasmic Ca2+ signaling through ryanodine receptor-dependent pathway, and enhanced the subsequent cAMP responsive element binding protein activity, matrix metalloproteinase-2 (MMP-2) gene expression, and endothelial tube forming. The abundance of mutated STIM1 and the MMP-2 expression were higher in native human umbilical vein endothelial cells of patients with gestational hypertension than controls and were significantly correlated with blood pressure. These findings broaden our understanding about structure–function bias of STIM1 and offer unique insights into its application in nucleoplasmic Ca2+, MMP-2 expression, endothelial dysfunction, and pathophysiological mechanism(s) of gestational hypertension.

Abstract 12665: Remodeling of Nuclear Ca 2+ Disposition in Atrial Fibrillation Associated Remodeling

Introduction: Atrial fibrillation (AF) induces substantial self-promoting remodeling related to the rapid atrial rate. There is evidence that changes in nuclear Ca 2+ content alter ion channel gene expression and thereby effect electrophysiological changes that increase AF vulnerability and maintenance. However, the ways in which AF affects atrial cardiomyocyte (ACM) nuclear Ca 2+ disposition are unknown. Here, we studied the effects of AF-related remodeling on ACM nuclear structure and Ca 2+ handling. Methods and Results: ACMs were isolated from control (CTL) dogs and dogs subjected to 1 week of AF maintained by atrial tachypacing at 600 bpm (n=7, 6 respectively). We used nuclear imaging with Fluo-5N AM and nucleoplasmic and cytoplasmic calcium transient (CaT) recordings from intact ACMs loaded with Fluo-4 AM. AF significantly decreased the number of nuclear invaginations (NIV) normalized to nuclear envelope (NE) circumference, and increased nuclear length and perimeter in ACMs (Figure A). Diastolic [Ca 2+ ] nuc and [Ca 2+ ] cyto increased in AF vs CTL (Figure B, by 110%*, 89%* respectively, *P<0.05). Nucleoplasmic Ca 2+ transient amplitude decreased significantly with AF, but simultaneously measured cytoplasmic transient amplitude did not change (Figure C). Relaxation time from peak of the Ca 2+ transient to 50% decline (TD 50 ) of nucleoplasmic and cytoplasmic CaTs were significantly prolonged in AF. The addition of a physiological nuclear Ca 2+ transient enhancer, endothelin (200 nM), increased diastolic [Ca 2+ ] nuc and [Ca 2+ ] cyto in both CTL (by 25±6%, 21±6%) and AF (by 40±4%, 31±2 ) ACMs. The IP3 receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 5 μM) abolished endothelin effects on [Ca 2+ ] nuc . Conclusions: AF-related remodeling alters atrial cardiomyocyte nuclear structure and nucleoplasmic [Ca 2+ ] handling. These alterations may play an important role in associated gene-expression changes that produce the AF substrate.

Caged ligands to study the role of intracellular GPCRs

In addition to cell surface membranes, numerous G protein–coupled receptors (GPCRs) are located on intracellular membranes including the nuclear envelope. Although the role of numerous GPCRs at the cell surface has been well characterized, the physiological function of these same receptors located on intracellular membranes remains to be determined. Here, we employ a novel caged Ang-II analog, cAng-II, to compare the effects of the activation of cell surface versus intracellular angiotensin receptors in intact cardiomyocytes. When added extracellularly to HEK 293 cells, Ang-II and photolysed cAng-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R). In contrast unphotolysed cAng-II did not. Cellular uptake of cAng-II was 6-fold greater than that of Ang-II and comparable to the HIV TAT(48–60) peptide. Intracellular photolysis of cAng-II induced an increase in nucleoplasmic Ca2+ ([Ca2+]n) that was greater than that induced by extracellular application of Ang-II. We conclude that cell-permeable ligands that can access intracellular GPCRs may evoke responses distinct from those with access restricted to the same receptor located on the cell surface.

Photoreleasable ligands to study intracrine angiotensin II signalling.

The renin-angiotensin system plays a key role in cardiovascular physiology and its overactivation has been implicated in the pathogenesis of several major cardiovascular diseases. There is growing evidence that angiotensin II (Ang-II) may function as an intracellular peptide to activate intracellular/nuclear receptors and their downstream signalling effectors independently of cell surface receptors. Current methods used to study intracrine Ang-II signalling are limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we present novel photoreleasable Ang-II analogues used to probe intracellular actions with spatial and temporal precision. The photorelease of intracellular Ang-II causes nuclear and cytosolic calcium mobilization and initiates the de novo synthesis of RNA in cardiac cells, demonstrating the application of the method. Several lines of evidence suggest that intracellular angiotensin II (Ang-II) contributes to the regulation of cardiac contractility, renal salt reabsorption, vascular tone and metabolism; however, work on intracrine Ang-II signalling has been limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we aimed to synthesize and characterize cell-permeant Ang-II analogues that are inactive without uncaging, but release active Ang-II upon exposure to a flash of UV-light, and act as novel tools for use in the study of intracrine Ang-II physiology. We prepared three novel caged Ang-II analogues, [Tyr(DMNB)(4)]Ang-II, Ang-II-ODMNB and [Tyr(DMNB)(4)]Ang-II-ODMNB, based upon the incorporation of the photolabile moiety 4,5-dimethoxy-2-nitrobenzyl (DMNB). Compared to Ang-II, the caged Ang-II analogues showed 2-3 orders of magnitude reduced affinity toward both angiotensin type-1 (AT1R) and type-2 (AT2R) receptors in competition binding assays, and greatly-reduced potency in contraction assays of rat thoracic aorta. After receiving UV-irradiation, all three caged Ang-II analogues released Ang-II and potently induced the contraction of rat thoracic aorta. [Tyr(DMNB)(4)]Ang-II showed the most rapid photolysis upon UV-irradiation and was the focus of subsequent characterization. Whereas Ang-II and photolysed [Tyr(DMNB)(4)]Ang-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R), caged [Tyr(DMNB)(4)]Ang-II did not. Cellular uptake of [Tyr(DMNB)(4)]Ang-II was 4-fold greater than that of Ang-II and significantly greater than uptake driven by the positive-control HIV TAT(48-60) peptide. Intracellular photolysis of [Tyr(DMNB)(4)]Ang-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n), and initiated 18S rRNA and nuclear factor kappa B mRNA synthesis in adult cardiac cells. We conclude that caged Ang-II analogues represent powerful new tools for use in the selective study of intracrine signalling via Ang-II.

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.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

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.

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.