{"content"=>"Phospholamban regulates nuclear Ca stores and inositol 1,4,5-trisphosphate mediated nuclear Ca cycling in cardiomyocytes.", "sup"=>[{"content"=>"2+"}, {"content"=>"2+"}]} (original) (raw)
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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.
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.
Nuclear Ca 2+ regulates cardiomyocyte function
Cell Calcium, 2008
In the heart, cytosolic Ca 2+ signals are well-characterized events that participate in the activation of cell contraction. In contrast, nuclear Ca 2+ contribution to cardiomyocyte function remains elusive. Here, we examined functional consequences of buffering nuclear Ca 2+ in neonatal cardiomyocytes. We report that cardiomyocytes contain a nucleoplasmic reticulum, which expresses both ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (InsP 3 R), providing a possible way for active regulation of nuclear Ca 2+ . Adenovirus constructs encoding the Ca 2+ buffer protein parvalbumin were targeted to the nucleus with a nuclear localization signal (Ad-PV-NLS) or to the cytoplasm with a nuclear exclusion signal (Ad-PV-NES). A decrease in the amplitude of global Ca 2+ transients and RyR-II expression, as well as an increase in cell beating rate were observed in Ad-PV-NES and Ad-PV-NLS cells. When nuclear Ca 2+ buffering was imposed nuclear enlargement, increased calcineurin expression, NFAT translocation to the nucleus and subcellular redistribution of atrial natriuretic peptide were observed. Furthermore, prolongation of action potential duration occurred in adult ventricular myocytes. These results suggest that nuclear Ca 2+ levels underlie the regulation of specific protein targets ଝ This work is attributed to Federal University of Minas Gerais.
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
In Situ Calibration of Nucleoplasmic versus Cytoplasmic Ca2+ Concentration in Adult Cardiomyocytes
Biophysical Journal, 2011
Quantification of subcellularly resolved Ca 2þ signals in cardiomyocytes is essential for understanding Ca 2þ fluxes in excitation-contraction and excitation-transcription coupling. The properties of fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca 2þ -dependent fluorescence changes into [Ca 2þ ] changes. Therefore, we determined the in situ characteristics of a frequently used Ca 2þ indicator, Fluo-4, and a ratiometric Ca 2þ indicator, Asante Calcium Red, and evaluated their use for reporting and quantifying cytoplasmic and nucleoplasmic Ca 2þ signals in isolated cardiomyocytes. Ca 2þ calibration curves revealed significant differences in the apparent Ca 2þ dissociation constants of Fluo-4 and Asante Calcium Red between cytoplasm and nucleoplasm. These parameters were used for transformation of fluorescence into nucleoplasmic and cytoplasmic [Ca 2þ ]. Resting and diastolic [Ca 2þ ] were always higher in the nucleoplasm. Systolic [Ca 2þ ] was usually higher in the cytoplasm, but some cells (15%) exhibited higher systolic [Ca 2þ ] in the nucleoplasm. Ca 2þ store depletion or blockade of Ca 2þ leak pathways eliminated the resting [Ca 2þ ] gradient between nucleoplasm and cytoplasm, whereas inhibition of inositol 1,4,5-trisphosphate receptors by 2-APB reversed it. The results suggest the presence of significant nucleoplasmic-to-cytoplasmic [Ca 2þ ] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca 2þ ] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca 2þ release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca 2þ stores.
Biochemistry, 2002
Alterations in expression levels of phospholamban (PLB) relative to the sarcoplasmic reticulum (SR) Ca-ATPase have been suggested to underlie defects of calcium regulation in the failing heart and other cardiac pathologies. To understand how variation in PLB expression relative to that of the Ca-ATPase can modulate calcium transport, we have investigated the inhibition of the Ca-ATPase by PLB in native SR membranes from slow-twitch skeletal and cardiac muscle and in reconstituted proteoliposomes. Quantitative immunoblotting in combination with affinity-purified protein standards was used to measure protein concentrations of PLB and of the Ca-ATPase. Functional inhibition of the Ca-ATPase was determined from both the calcium concentrations for half-maximal activation (Ca 1/2 ) and the shift in the calcium concentrations following release of PLB inhibition (i.e., ∆Ca 1/2 ) by incubation with monoclonal antibodies against PLB, which are equivalent to phosphorylation of PLB by cAMP-dependent protein kinase. We report that equivalent levels of PLB inhibition and antibody-induced activation (∆Ca 1/2 ) 0.25 ( 0.02 µM) are observed in SR membranes from slow-twitch skeletal and cardiac muscle, where molar stoichiometries of PLB expressed per Ca-ATPase vary, respectively, from 0.9 ( 0.1 to 4.1 ( 0.8. Similar levels of inhibition to those observed in isolated SR vesicles were observed using reconstituted proteoliposomes following co-reconstitution of affinity-purified Ca-ATPase with PLB. These results indicate that total expression levels of one PLB per Ca-ATPase result in full inhibition of the Ca-ATPase and, based on the measured K D (140 ( 30 µM), suggests one PLB complexed with two Ca-ATPase molecules is sufficient for full inhibition of activity. Therefore, the excess PLB expressed in the heart over that required for inhibition suggests a capability for graded responses of the Ca-ATPase activity to endogenous kinases and phosphatases that modulate the level of phosphorylation necessary to relieve inhibition of the Ca-ATPase by PLB.
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.