Cardiac Myocytes from Newborn Mice (original) (raw)
Nuclear and cytosolic calcium are regulated independently
Proceedings of the National Academy of Sciences, 2003
Nuclear calcium (Ca 2؉ ) regulates a number of important cellular processes, including gene transcription, growth, and apoptosis. However, it is unclear whether Ca 2؉ signaling is regulated differently in the nucleus and cytosol. To investigate this possibility, we examined subcellular mechanisms of Ca 2؉ release in the HepG2 liver cell line. The type II isoform of the inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R) was expressed to a similar extent in the endoplasmic reticulum and nucleus, whereas the type III InsP 3R was concentrated in the endoplasmic reticulum, and the type I isoform was not expressed. Ca 2؉ signals induced by low InsP3 concentrations started earlier or were larger in the nucleus than in the cytosol, indicating higher sensitivity of nuclear Ca 2؉ stores for InsP3. Nuclear InsP3R channels were active at lower InsP 3 concentrations than InsP3R from cytosol. Enriched expression of type II InsP 3R in the nucleus results in greater sensitivity of the nucleus to InsP 3, thus providing a mechanism for independent regulation of Ca 2؉ -dependent processes in this cellular compartment.
Journal of Clinical Investigation, 2006
Previous work showed that calmodulin (CaM) and Ca 2+ -CaM-dependent protein kinase II (CaMKII) are somehow involved in cardiac hypertrophic signaling, that inositol 1,4,5-trisphosphate receptors (InsP 3 Rs) in ventricular myocytes are mainly in the nuclear envelope, where they associate with CaMKII, and that class II histone deacetylases (e.g., HDAC5) suppress hypertrophic gene transcription. Furthermore, HDAC phosphorylation in response to neurohumoral stimuli that induce hypertrophy, such as endothelin-1 (ET-1), activates HDAC nuclear export, thereby regulating cardiac myocyte transcription. Here we demonstrate a detailed mechanistic convergence of these 3 issues in adult ventricular myocytes. We show that ET-1, which activates plasmalemmal G protein-coupled receptors and InsP 3 production, elicits local nuclear envelope Ca 2+ release via InsP 3 R. This local Ca 2+ release activates nuclear CaMKII, which triggers HDAC5 phosphorylation and nuclear export (derepressing transcription). Remarkably, this Ca 2+ -dependent pathway cannot be activated by the global Ca 2+ transients that cause contraction at each heartbeat. This novel local Ca 2+ signaling in excitation-transcription coupling is analogous to but separate (and insulated) from that involved in excitation-contraction coupling. Thus, myocytes can distinguish simultaneous local and global Ca 2+ signals involved in contractile activation from those targeting gene expression.
Cell Calcium, 2008
Dynamic nuclear Ca 2+ signals play pivotal roles in diverse cellular functions including gene transcription, cell growth, differentiation, and apoptosis. Here we report a novel nuclear Ca 2+ regulatory mechanism mediated by inositol 1,4,5-trisphosphate receptors (IP 3 Rs) around the nucleus in developing cardiac myocytes. Activation of IP 3 Rs by ␣ 1 -adrenergic receptor (␣ 1 AR) stimulation or by IP 3 application (in saponinpermeabilized cells) increases Ca 2+ spark frequency preferentially in the region around the nucleus in neonatal rat ventricular myocytes. A nuclear enrichment of IP 3 R distribution supports the higher responsiveness of Ca 2+ release in this particular region. Strikingly, we observed "nuclear Ca 2+ waves" that engulf the entire nucleus without spreading into the bulk cytosol. ␣ 1 AR stimulation enhances the occurrence of nuclear Ca 2+ waves and confers them the ability to trigger cytosolic Ca 2+ waves via IP 3 R-dependent pathways. This finding accounts, at least partly, for a profound frequency-dependent modulation of global Ca 2+ oscillations during ␣ 1 AR stimulation. Thus, IP 3 R-mediated Ca 2+ waves traveling in the nuclear region provide active, autonomous regulation of nuclear Ca 2+ signaling, which provides for not only the local signal transduction, but also a pacemaker to drive global Ca 2+ transient in the context of ␣ 1 AR stimulation in developing cardiac myocytes.
Channels, 2010
C ardiac hypertrophy is associated with profound remodeling of Ca 2+ signaling pathways. During the early, compensated stages of hypertrophy, Ca 2+ fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca 2+ homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca 2+ release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca 2+ store are primarily responsible for the Ca 2+ flux that induces myocyte contraction, a role for Ca 2+ release via the inositol 1,4,5-trisphosphate receptor (InsP 3 R) in cardiac physiology has also emerged. Specifically, InsP 3 -induced Ca 2+ signals generated following myocyte stimulation with an InsP 3 -generating agonist (e.g., endothelin, ET-1), lead to modulation of Ca 2+ signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca 2+ release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP 3 R (InsP 3 R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca 2+ release via InsP 3 Rs serves to sensitize neighboring RyRs causing an augmentation of Ca 2+ fluxes during ECC as well as an increase in non-triggered Ca 2+ release events. This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Calcium sensing receptor regulates cardiomyocyte function through nuclear calcium
Cell Biology International, 2012
Nuclear Ca 2+ plays a pivotal role in the regulation of gene expression. IP 3 (inositol-1,4,5-trisphosphate) is an important regulator of nuclear Ca 2+. We hypothesized that the CaR (calcium sensing receptor) stimulates nuclear Ca 2+ release through IICR (IP 3-induced calcium release) from perinuclear stores. Spontaneous Ca 2+ oscillations and the spark frequency of nuclear Ca 2+ were measured simultaneously in NRVMs (neonatal rat ventricular myocytes) using confocal imaging. CaR-induced nuclear Ca 2+ release through IICR was abolished by inhibition of CaR and IP 3 Rs (IP 3 receptors). However, no effect on the inhibition of RyRs (ryanodine receptors) was detected. The results suggest that CaR specifically modulates nuclear Ca 2+ signalling through the IP 3 R pathway. Interestingly, nuclear Ca 2+ was released from perinuclear stores by CaR activator-induced cardiomyocyte hypertrophy through the Ca 2+-dependent phosphatase CaN (calcineurin)/ NFAT (nuclear factor of activated T-cells) pathway. We have also demonstrated that the activation of the CaR increased the NRVM protein content, enlarged cell size and stimulated CaN expression and NFAT nuclear translocation in NRVMs. Thus, CaR enhances the nuclear Ca 2+ transient in NRVMs by increasing fractional Ca 2+ release from perinuclear stores, which is involved in cardiac hypertrophy through the CaN/NFAT pathway.
Journal of Molecular and Cellular Cardiology, 2012
Inositol 1,4,5-trisphosphate (InsP 3 R)-mediated Ca 2+ signaling is a major pathway regulating multiple cellular functions in excitable and non-excitable cells. Although InsP 3-mediated Ca 2+ signaling has been extensively described, its influence on ventricular myocardium activity has not been addressed in contracting hearts at the whole-organ level. In this work, InsP 3-sensitive intracellular Ca 2+ signals were studied in intact hearts using laser scanning confocal microscopy and pulsed local-field fluorescence microscopy. Intracellular [InsP 3 ] was rapidly increased by UV flash photolysis of membrane-permeant caged InsP 3. Our results indicate that the basal [Ca 2+ ] increased after the flash photolysis of caged InsP 3 without affecting the action potential (AP)-induced Ca 2+ transients. The amplitude of the basal [Ca 2+ ] elevation depended on the intracellular [InsP 3 ] reached after the UV flash. Pretreatment with ryanodine failed to abolish the InsP 3-induced Ca 2+ release (IICR), indicating that this response was not mediated by ryanodine receptors (RyR). Thapsigargin prevented Ca 2+ release from both RyR-and InsP 3 R-containing Ca 2+ stores, suggesting that these pools have similar Ca 2+ reuptake mechanisms. These results were reproduced in acutely isolated cells where photorelease of InsP 3 was able to induce changes in endothelial cells but not in AP-induced transients from cardiomyocytes. Taken together, these results suggest that IICR does not directly regulate cardiac excitationcontraction coupling. To our knowledge, this is the first demonstration of IICR in intact hearts. Consequently, our work provides a reference framework of the spatiotemporal attributes of the IICR under physiological conditions.
Canadian Journal of Physiology and Pharmacology, 2006
Cytosolic Ca 2+ is a versatile secondary messenger that regulates a wide range of cellular activities. In the past decade, evidence has accumulated that free Ca 2+ within the nucleus also plays an important messenger function. Here we review the mechanisms and effects of Ca 2+ signals within the nucleus. In particular, evidence is reviewed that the nucleus contains the machinery necessary for production of inositol 1,4,5-trisphosphate and for inositol 1,4,5-trisphosphate receptor-mediated Ca 2+ release. The role of Ca 2+ signals within the nucleus is discussed including regulation of such critical cell functions as gene expression, activation of kinases, and permeability of nuclear pores.
STEM CELLS, 2008
On the basis of previous findings suggesting that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the sarcoplasmic reticulum Ca 2؉ -induced release of calcium machinery is either absent or immature, in the present study we tested the hypothesis that hESC-CM contain fully functional 1,4,5-inositol trisphosphate (1,4,5-IP 3 )-operated intracellular Ca 2؉ ([Ca 2؉ ] i ) stores that can be mobilized upon appropriate physiological stimuli. To test this hypothesis we investigated the effects of angiotensin-II (AT-II) and endothelin-1 (ET-1), which activate the 1,4,5-IP 3 pathway, on [Ca 2؉ ] i transients and contractions in beating clusters of hESC-CM. Our major findings were that in paced hESC-CM both AT-II and ET-1 (10 ؊9 to 10 ؊7 M) increased the contraction amplitude and the maximal rates of contraction and relaxation. In addition, AT-II (10 ؊9 to 10 ؊7 M) increased the [Ca 2؉ ] i transient amplitude. The involvement of 1,4,5-IP 3dependent intracellular Ca 2؉ release in the inotropic effect of AT-II was supported by the findings that (a) hESC-CM express AT-II, ET-1, and 1,4,5-IP 3 receptors determined by immunofluorescence staining, and (b) the effects of AT-II were blocked by 2 M 2-aminoethoxyphenyl borate (a 1,4,5-IP 3 receptor blocker) and U73122 (a phospholipase C blocker). In conclusion, these findings demonstrate for the first time that hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP 3 -operated releasable Ca 2؉ stores.
Nuclear calcium signaling: a cell within a cell
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 2009
Calcium (Ca2+) is a versatile second messenger that regulates a wide range of cellular functions. Although it is not established how a single second messenger coordinates diverse effects within a cell, there is increasing evidence that the spatial patterns of Ca2+ signals may determine their specificity. Ca2+ signaling patterns can vary in different regions of the cell and Ca2+ signals in nuclear and cytoplasmic compartments have been reported to occur independently. No general paradigm has been established yet to explain whether, how, or when Ca2+ signals are initiated within the nucleus or their function. Here we highlight that receptor tyrosine kinases rapidly translocate to the nucleus. Ca2+ signals that are induced by growth factors result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. This novel signaling mechanism may be responsible for growth factor effects on cell prolifer...
Circulation Research, 2013
Cellular Biology T hroughout the past 2 decades, intense research has linked nuclear Ca 2+ signals to a wide range of physiological and pathological cellular responses. 1,2 Although nuclear Ca 2+ is essential for processes such as nuclear transport, chromatin condensation, and the activation of several transcription factors, 3-6 the origin of the nuclear Ca 2+ signal is still controversial. Some authors suggest that nuclear Ca 2+ signals can result from Ca 2+ release that initiates in the cytosol and then propagates to the nucleus, 7,8 whereas others suggest that it occurs independently of cytosolic Ca 2+ release. 9,10 Consistent with the latter notion, we have previously reported that stimulation of the insulin-like growth factor 1 receptor (IGF-1R) triggers a rapid inositol 1,4,5-trisphosphate (IP 3
An integrated mechanism of cardiomyocyte nuclear Ca(2+) signaling
Journal of molecular and cellular cardiology, 2014
In cardiomyocytes, Ca(2+) plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca(2+) within subcellular microdomains: transcription is regulated by Ca(2+) release within nuclear microdomains, and excitation-contraction coupling is regulated by cytosolic Ca(2+). Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca(2+) signals. However, signaling pathways coupling surface receptor activation to nuclear Ca(2+) release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca(2+) signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the n...
Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum
Nature Cell Biology, 2003
Calcium is a second messenger in virtually all cells and tissues 1 . Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP 3 ) receptors, and the nuclear component of InsP 3mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP 3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus.
The InsP3 receptor and intracellular Ca2+ signaling
Current Opinion in Neurobiology, 1997
The inositol 1,4,5trisphosphate receptor (InsPsR) is a ligand-gated Ca2+-release channel on intracellular Ca*+ store sites (such as the endoplasmic reticulum), and plays an important role in intracellular Ca*+ signaling in a wide variety of cell types. Recent studies have shown that binding of inositol 1,4,5-trisphosphate (InsPs) to lnsPsR isoforms is differentially regulated by Ca*+, and that InsPsR functions are finely regulated by phosphorylation via tyrosine kinases and protein kinase C, by dephosphorylation via calcineurin, and by binding to FKBP (FK506-binding protein). In addition, transient receptor potential (TRP) and TRP-like proteins appear to couple conformationally with the InsPsR for capacitative Ca*+ entry. The importance of InsPsR signaling in neuronal function has been demonstrated by gene targeting in mice and by studies of T-cell receptor signaling, apoptosis, meiotic maturation, and cytokinesis. Addresses Discs large FKBP FK506-binding protein IICR InsPainduced Ca*+ release inad inactivation no after potential D InsP, inositol 1,4,5trisphosphate InsPsR InsPs receptor opt opisthotonos PCR polymerase chain reaction PDZ PSD-95, Dig. ZO-1 PKC protein kinase C PLC phospholipase C PSD-95 postsynaptic density protein of 95 kDa RyR ryanodine receptor TCR T-cell receptor TRP transient receptor potential TRPL TRP-like zo-1 zona occludens 1 2. Furuichi T, Kohda K, Miyawaki A, Mikoshiba K: Intracellular channels. Curr Opin Neurobiol 1994, 4:294-303. 3. Ross CA, Danoff SK, Schell MJ, Snyder SH: Three additional inositol 1.4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues. Proc Nat/ Acad Sci USA 1992,89:4265-4269. 4. De Smedt H, Missieaen L, Parys JB, Bootman MD, Mertens L, Van Den Bosch L, Casteels R: Determination of relative amounts of inositol trisphosohate receptor mRNA isoforms by ratio polymerese chain reaction. I Biol C/rem 1994, 269:21691-21698. 5. Parys JB, Missiaen L, De Smedt H, Sienaert I, Casteels R: Mechanisms responsible for quantel Cal+ release from inositol triophosphate-sensitive celcium stores. fur J Physioll996, 432:359-367. 6. Nahorski SR, Potter BL: Molecular recognition of inositol polyphosphates by intracellular receptors and metabolic enxymes. fiends Pharmacol Sci 1969, IO:1 39-l 44. Z 8. 9. 10. 11. 12. . . Wilcox RA, Challiss RAJ, Traynor JR, Fauq AH, Ognayanov VI, Korikowski AP, Nahorski SR: Molecular recognition at the myo-inositol 1,4,5+isphosphate receptor. J Biol Chem 1994, 269:26615-26821. O'Rouke F, Feinstein MB: The inositol 1 ,4.5-trisphosphate receptor binding sites of platelet membranes. Biochem J 1990, 267:297-302. Mignery GA, SiJdhof TC: The ligand binding site and transduction mechanism in the inositol 1,4,5+isphosphate receptor. EM60 J 1990, 9:3893-3696. Miyawaki A, Furuichi T, Ryou Y, Yoshikawa S, Nakagawa T, Saitoh T, Mikoshiba K: Structure-function relationships of the mouse inositol 1,4,6-trisphosphate receptor. Proc fiat/ Acad Sci USA 1991, 96:491 l-491 5. Newton CL, Mignery GA, Siidhof TC: Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5trisphosphate (InsPs) receptors with distinct affinities for InsPs. J Biol Chem 1994, 26826616-28619. Brillantes A-M, Ondrias K, Scott A, Kobrinsky E, Ondriasova E, Moschella MC, Jayaraman T, Landers M, Ehrfich BE, Marks AR: Stebilization of celcium releese channel (ryenodine receptor) function by FK506-binding protein. Cell 1994, 77:513-523. Timerrnan AP, Wiederrecht G, Marcy A, Fleischer S: Characterization of an exchange reaction between soluble FKBP-12 and the FKBP/ryanodine receptor complex. J Viol Chem 1995, 270:2451-2459. Cameron AM, Steiner JP, Roskarns Al, Ali SM, Ronnett GV, Snyder SH: Calcineurin associated with the inositol 1,4,5trisphosphate receptor-FKBP12 complex modulates Ca*+ flux Cell 1995, 83~463-472.
Regulation of Ins(1,4,5)P3 3-kinases by calcium and localization in cells
Ins(1,4,5)P 3 3-kinases (IP 3 Ks) are a group of calmodulin-regulated inositol polyphosphate kinases (IPKs) that convert the second messenger Ins(1,4,5)P 3 into Ins(1,3,4,5)P 4 . However, what they contribute to the complexities of Ca 2+ signalling, and how, is still not fully understood. In this study, we have used a simple Ca 2+ imaging assay to compare the abilities of various Ins(1,4,5)P 3 metabolizing enzymes to regulate a maximal histaminestimulated Ca 2+ signal in HeLa cells. Using transient transfection, we over-expressed GFPtagged versions of all three mammalian IP 3 K isoforms, including mutants with disrupted cellular localization or CaM regulation, and then imaged the Ca 2+ release stimulated by 100µM histamine. Both localization to the Factin cytoskeleton and CaM regulation enhance the efficiency of mammalian IP 3 Ks to dampen the Ins(1,4,5)P 3 -mediated Ca 2+ signals. We also compared the effects of the these IP 3 Ks to other enzymes that metabolize Ins(1,4,5)P 3 , including the Type I Ins(1,4,5)P 3 5-phosphatase, in both membrane-targeted and soluble forms, the human InsP 3 multikinase (IPMK), and the two isoforms of IP 3 K found in Drosophila. All reduce the Ca 2+ signal, but to varying degrees. We demonstrate that the activity of only one of two IP 3 K isoforms from Drosophila is positively regulated by CaM, and that neither isoform associates with the cytoskeleton. Together the data suggest that IP 3 Ks evolved to regulate kinetic and spatial aspects of Ins(1,4,5)P 3 signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.
Biological Research, 2006
Nuclear calcium appears to have an important role in the regulation of gene expression in many cells, but the mechanisms involved in controlling nuclear Ca 2+ signaling are controversial and still poorly understood. We have described the presence of inositol 1,4,5 trisphosphate (IP 3 ) receptors in the nuclei of skeletal muscle cells. Now, we have characterized the properties of the IP 3 receptors channels present in the nuclei of the 1B5 cell line, which do not express any isoforms of the ryanodine receptor. Immunocytochemistry of isolated nuclei confirmed the presence of IP 3 R in the nuclear envelope and fluorescence measurements in nuclei suspensions allowed us to document ATP-dependent calcium loading by the nucleus and its release upon IP 3 addition. Patch clamp of nuclear membranes was performed, and single-channel activity recorded was dependent on the presence of IP 3 in the pipette; single-channel conductance was in the range reported in the literature for these channels, and the open probability was shown to be dependent on IP 3 concentration. The presence of functional IP 3 receptors in the nuclear envelope membrane is likely to have an important function in the regulation of nucleoplasmic calcium concentration and consequently in the regulation of transcription in muscle cells.