1Research Services, Edward Hines Jr. Department of Veterans Affairs Hospital, Hines 60141; and 2Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612
Submitted 6 January 2004 ; accepted in final form 14 June 2004
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ABSTRACT |
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calcium release channels; cell signaling; calcium handling; inositol trisphosphate receptors
The idea of InsP3R involvement in cell growth is supported by experiments performed by other investigators in which long-term activation of signaling pathways that generate InsP3 results in expression of genes involved in cellular hypertrophy (3, 10, 14, 55). However, how cardiac cells discriminate between the InsP3-induced changes in calcium and the large and cyclic fluctuations of calcium during excitation-contraction coupling is unknown. Furthermore, it is also unknown how InsP3Rs may alter expression of genes during cell growth. The phenomenon of slow waves of calcium or calcium oscillations promoting expression of hypertrophic genes in other systems (8, 23) does not fit with the normal functioning of cardiac cells because they experience rhythmic elevations of calcium at a much faster rate. Thus cardiac myocytes must tightly regulate the factors that alter gene expression. A plausible explanation is that calcium release by InsP3Rs in cardiac myocytes occurs at discrete sites where it would have a direct effect on gene expression. To test this hypothesis, we investigated the localization of InsP3Rs, their ability to release calcium, and a possible regulatory function of these receptors on the expression of some of the hypertrophic marker genes.
The results reveal the previously unidentified presence of type 2 InsP3Rs associated with the nuclear region and in striations in cardiac myocytes. In addition, the data provide strong evidence in favor of type 2 InsP3R involvement in intracellular signaling and promotion of cardiac cell growth.
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MATERIALS AND METHODS |
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Confocal microscopy. Single cells were examined for InsP3R localization and changes in calcium concentration with a laser scanning confocal system (Bio-Rad Radiance 2100) attached to an inverted microscope (Nikon Eclipse TE300) with a x60, 1.2 numerical aperture water-immersion objective.
To detect InsP3Rs, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100. Cells were then incubated in 10% goat serum in PBS for 23 h to block nonspecific labeling. Primary antibodies were diluted in PBS plus 0.1% Triton X-100. The anti-type 1 InsP3R antibody (Affinity BioReagents) was used at a 1:250 dilution. For detection of type 2 InsP3R we used an antibody against the amino terminal (Affinity BioReagents) or the carboxy terminal (Santa Cruz Biotechnology), both at a 1:200 dilution. The anti-type 3 InsP3R antibody (Sigma-Aldrich) was also used at a 1:200 dilution. The secondary antibody carried an FITC derivative (Alexa Fluor 488; Molecular Probes) and was used at a 1:750 dilution. Cells were incubated for 2 h in the primary antibody and 2 h in the secondary antibody. After incubation with the secondary antibody, cells were rinsed twice with PBS containing 0.2% RNase. To label the nuclei, cells were exposed to 3 µM TO-PRO-3 (Molecular Probes), which binds to double-stranded nucleic acid. In some cases, actin filaments were also labeled with rhodamine phalloidin (1:1,000 dilution; Molecular Probes). Images of 512 x 512 pixels were collected in xy with a pinhole size of 1.0 Airy Disk. Images were then imported into Adobe Photoshop for cropping.
Intracellular calcium signals were detected from cells permeabilized by exposure to 0.01% saponin for 2 min (32). The permeabilization solution contained (in mM) 100 K-aspartate, 20 KCl, 3 MgATP, 0.81 MgCl2, 0.5 EGTA, 10 phosphocreatine, and 20 HEPES, with 5 U/ml creatine phosphokinase, pH 7.2. The control test solution was similar to the permeabilization solution except that it contained 0.114 CaCl2, 0.100 tetracaine, and 0.03 K5-fluo 4 and saponin was omitted. The free calcium and magnesium concentrations in these solutions were 100 nM and 1 mM, respectively. To activate InsP3Rs, 50 nM Adenophostin A (Calbiochem; Ref. 50) was added to the test solution. To block InsP3Rs, we used 2-aminoethoxydiphenyl borate (2-APB; Ref. 33) or xestospongin C (XeC; Ref. 16) (Calbiochem).
Calcium events were recorded in line scan mode as we previously described (46). Scans consisted of 5,000 lines of 512 pixels each. The pixel size was 0.31 µm. Events were detected and measured with an automated algorithm as described by Cheng et al. (9). The fluorescence intensity of the events (F) was normalized to the baseline fluorescence (Fo) obtained by measuring the fluorescence over time when no events were detected. The normalized fluorescence is thus reported as F/Fo.
Alexa Fluor 488 and fluo 4 were excited with the 488-nm line of a krypton/argon laser. Emission was measured at wavelengths of 515 ± 30 nm. Rhodamine phalloidin was excited with the 568-nm line of the laser, and its emission was measured at 600 ± 40 nm. TO-PRO-3 was excited at 637 nm, and emission was measured at wavelengths>660 nm.
Measurement of gene expression.
Total RNA was isolated from control or treated cells from the same culture following the manufacturer's instructions (Qiagen). RNA (0.2 µg) was mixed with 100 pmol of random hexamer primers (Roche) and heated to 70°C for 2 min. After denaturation, the mix was added to the reverse transcription cocktail containing 200 U of Moloney murine leukemia virus reverse transcriptase and incubated for 1 h at 37°C. Gene-specific PCR was performed using 3 µl of the amplified cDNA (10 µl when measuring L-type calcium channel subunit 2
1-C) in a 500-µl volume with 1x PCR buffer, 1.5 mM MgCl2, 1 mM dNTP, 0.5 U of Taq DNA polymerase (Eppendorf), and 10 pmol of each primer. The gene of interest and GAPDH were simultaneously amplified in a single PCR reaction tube to allow direct comparison of the relative amounts of message. PCR amplification was performed within the linear range as we previously determined for these genes (2).
Western blotting. Cellular fractions were separated from isolated neonatal cells with a subcellular proteome extraction kit (Calbiochem) following the manufacturer's instructions for cells in suspension. The four fractions obtained (cytosol, membrane/organelle, nucleus, and cytoskeleton) were used immediately or stored at 80°C until use. Proteins were separated by SDS-polyacrylamide gel electrophoresis using 7.5% gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the amino-terminal anti-type 2 InsP3R primary antibody used for immunolocalization of InsP3Rs with confocal microscopy at a 1:1,000 dilution. After washing and blocking, membranes were incubated with a secondary antibody conjugated to horseradish peroxidase at a 1:20,000 dilution. Antibody binding was detected with chemiluminescence by using the SuperSignal West Femto Detection Kit (Pierce) according to the manufacturer's instructions. To determine the purity of these fractions, blots were also examined with antibodies against specific proteins in each subcellular fraction: anti-calpain-1 (Affinity Bioreagents) for the cytosolic fraction, anti-sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a (Sigma) for the membrane/organelle fraction, anti-histone deacetylase 2 (Zymed Laboratories) for the nuclear fraction, and anti-desmin (Sigma) for the cytoskeleton fraction. These antibodies were also detected with chemiluminescence. The distributions of the signals in the cytosol, membrane/organelle, nucleus, and cytoskeleton fractions, respectively, for three determinations were as follows (means ± SE): calpain-1: 94 ± 2%, 4 ± 0.3%, 2 ± 0.6%, 0%; SERCA2a: 2 ± 0.6%, 95 ± 0.4%, 3 ± 0.9%, 0%; histone deacetylase 2: 0%, 1.7 ± 0.3, 98 ± 0.9, 0.7 ± 0.7%; and desmin: 3 ± 1.2%, 3.7 ± 0.3%, 3.3 ± 0.3%, 90 ± 0.9%.
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RESULTS |
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The images in Fig. 3 correspond to a representative cell labeled with the amino-terminal (first 4 rows) or the carboxy-terminal (last row) anti-type 2 InsP3R antibody that had only type 2 InsP3R staining associated with nuclear staining. The first four rows in Fig. 3 are a series of successive images taken from different planes of the cell at 0.5-µm increments, and they show a clearer relationship of the type 2 InsP3Rs with the nuclear staining. The last row in Fig. 3 was obtained with the carboxy-terminal antibody, and it demonstrates the same association of type 2 InsP3R with nuclear staining as that shown with the amino-terminal antibody. Thus the results obtained with antibodies to either the amino or the carboxy terminal of the type 2 InsP3R protein are indistinguishable. In addition, detection of type 2 InsP3R with the carboxy-terminal antibody suggests that the full-length protein is being expressed.
A low level of type 2 InsP3R staining was also detected throughout the cells in the form of discrete clusters. To eliminate the possibility of bleed-through of TO-PRO-3 staining or nonspecific background staining, control experiments were performed in which the primary antibody was omitted or preadsorbed with the peptide used to generate the antibody. Both controls gave identical results. Figure 2, i and j, shows the nuclei in the cells and the absence of type 2 InsP3R staining when the primary antibody was omitted. The consistent pattern of colocalization of type 2 InsP3Rs in the nuclear region in neonatal cardiac myocytes provides support for the putative involvement of these receptors in modulation of nuclear calcium and changes in gene expression.
Detection of type 2 InsP3Rs in cellular fractions by Western blot.
Isolated cardiocytes were suspended, fractionated, and used to determine the subcellular location of type 2 InsP3Rs. Proteins were electrophoresed on 7.5% SDS-polyacrylamide gels and transferred to PVDF membranes. Incubation of the membranes with the antibody directed against the amino terminal of type 2 InsP3Rs showed a single signal with a molecular mass of 260 kDa (Fig. 4), which corresponds to the expected molecular weight of the type 2 InsP3R (36). The immunoreactive signal was elicited in both the membrane/organelle and nuclear fractions. Interestingly, the signal intensity was stronger in the nuclear fraction than in the membrane/organelle fraction. There was no apparent signal in either the cytosolic or the cytoskeletal fraction. Detection of signals in the nuclear and membrane/organelle fractions agrees with the colocalization of type 2 InsP3Rs with the nuclear staining and rhodamine phalloidin staining, respectively, found by confocal microscopy. Furthermore, the strength of the signals seen in Western blots correlates with the fact that type 2 InsP3Rs are always found in association with nuclear staining but not with rhodamine phalloidin in neonatal cardiac cells.
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Potential modulation of gene expression by type 2 InsP3Rs.
To determine the role of calcium release in the nuclear domains, we measured gene expression of some of the molecules that are affected when signaling cascades involving InsP3Rs are activated, such as atrial natriuretic factor (ANF) and skeletal -actin (3, 13, 14, 48). Gene expression was measured with RT-PCR. The RNA message was normalized to expression of GAPDH measured concurrently with each gene examined, and results from test conditions were normalized to the paired controls. Equal amounts of RNA were used in each RT-PCR assay. Cardiac cells were exposed to the agonists endothelin-1 (1 µM) or phenylephrine (10 µM) for 4872 h to stimulate the hypertrophic response. Endothelin-1 and phenylephrine significantly increased the expression of both ANF and skeletal
-actin genes (Fig. 6). The average increase of ANF and skeletal
-actin message with endothelin-1 was 2.05 ± 0.3 (n = 9 cultures) and 1.87 ± 0.32 (n = 6 cultures) times over control conditions, respectively (P < 0.05). The corresponding average increase with phenylephrine was 1.90 ± 0.35 (n = 7 cultures) and 1.65 ± 0.28 (n = 6 cultures) times over control, respectively (P < 0.05). Although the expression of ANF and skeletal
-actin was significantly higher in the presence of the hypertrophic agonists than under control conditions, the increase is smaller than that found in previous studies using rat cardiac myocytes (3, 13, 14, 26), which most likely is a reflection of the difference in species (11, 45). Furthermore, it should be noted that hypertrophy of mouse myocytes in response to receptor activation in vitro has been seen by some investigators (27, 34, 37, 42, 52) but not by others (11, 45), probably reflecting differences in culture conditions. In our experiments, exposure of the myocytes to 2-APB (40 µM) or XeC (20 µM) resulted in a marked inhibition of expression of these two genes. It has been reported that 2-APB and XeC can affect the calcium transporters SERCA2 and RyR in addition to InsP3R and result in depletion of the endoplasmic reticulum in other cell types (6, 18, 22, 39, 41, 47). To determine whether the InsP3R antagonists had deleterious secondary effects in neonatal myocytes at the concentrations used here, we treated myocytes with thapsigargin (12 µM) to block SERCA2 and deplete the sarcoplasmic reticulum of calcium. However, these conditions were detrimental for the cells because they detached from the culture dishes and did not last for the duration of the experiment. We also exposed myocytes to tetracaine and found that it did not prevent the change in gene expression promoted by endothelin-1 or phenylephrine. Thus our results suggest that InsP3Rs mediate the increase in mRNA levels.
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DISCUSSION |
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The sole presence of type 2 InsP3Rs in neonatal mouse myocytes found here agrees with data obtained with isolated neonate rat ventricular myocytes in which type 2 InsP3R is the only isoform found with RT-PCR (40) and suggests that this isoform is a critical component of intracellular signaling and the expression of hypertrophic genes. It was suggested previously that type 1 InsP3Rs are involved in intercellular signaling because they have been identified in cardiac cells at the intercalated disk by immunogold electron microscopy (25) and in the sarcoplasmic reticulum of cells from the conduction system (17). Here we determine the localization of InsP3Rs and provide support for a regulatory role of type 2 InsP3Rs in gene expression in neonatal cardiac cells. Using two antibodies that have been shown to be specific for the type 2 InsP3R (7, 20, 28, 30, 43), we demonstrate with confocal microscopy that these receptors are preferentially localized in the region of the nucleus and less frequently in association with the sarcomeres. Analysis of type 2 InsP3R protein in subcellular fractions with SDS-PAGE and immunoblotting also demonstrates that type 2 InsP3Rs are found in the membrane/organelle and nuclear fractions, which supports the data obtained with confocal microscopy.
Although the goal of this study was to determine the localization and function of InsP3Rs in cardiac myocytes, the results supply additional and important information on the cell biology of neonatal mouse cardiac myocytes. The vast majority of studies involving signaling cascades in cardiac myocytes in vitro have been delineated by work done mostly with primary culture of neonatal rat ventricular cells. Even though rat cells have provided a wealth of valuable information, differences in signaling cascades between neonatal rat and neonatal mouse cardiac myocytes have been reported, most notably by Steinberg's (45) and Simpson's (11) labs. However, it should be noted, parenthetically, that there are also conflicting results between these two reports. For example, Sabri et al. (45) found that norepinephrine is ineffective in promoting hypertrophy and inositol phosphate accumulation in mouse myocytes. On the contrary, results from Deng et al. (11) showed that norepinephrine induced hypertrophy and that exposure to endothelin and phenylephrine promoted accumulation of inositol phosphates in mouse cells, which led them to conclude that aspects of acute signaling were intact in mouse myocytes. Our own experiments also show that the increase in ANF and skeletal -actin mRNA is considerably smaller than that observed in rat myocytes. Although there are differences between signaling in neonatal rat and mouse myocytes, mice have become the preferred animal model to study cardiac hypertrophy because of transgenic technology. Results obtained from adult mice are compared with those from the historically preferred model of neonatal rat cells in culture. Thus it is not surprising to find that a correlation does not always exist between the data obtained from in vivo and in vitro experiments or from adult and neonatal cells. This discrepancy underscores the importance of examining the signaling cascades in neonatal mouse myocytes. The results obtained here show that neonatal mouse myocytes do experience changes in expression of at least some of the genes involved in hypertrophy on exposure of the cells to endothelin-1 and phenylephrine. More work is needed to determine whether type 2 InsP3Rs modulate expression of other hypertrophic genes and to determine the distribution of InsP3Rs in adult ventricular myocytes.
Despite the additional effects that 2-APB and XeC can have on various calcium transporters, our results suggest that the action of these antagonists on InsP3R was mainly responsible for the blockade of the endothelin-1 and phenylephrine responses. In addition to preventing the effect of endothelin-1 and phenylephrine, the InsP3Rs blockers also decreased the levels of ANF and skeletal -actin in the absence of the agonists. These novel and exciting results suggest that InsP3Rs are important for maintaining basal expression of these two genes, which are involved in growth and development in cardiac myocytes, and support the idea that type 2 InsP3Rs are involved in the regulation of cellular responses such as growth and programmed cell death (19, 35, 44).
Although type 2 InsP3Rs have also been found associated with the nucleus of other cell types (28), our work is the first to show that this calcium release channel is localized in association with the nucleus and the sarcomeres of cardiac myocytes. Quite possibly, these receptors are embedded in the tubular membrane-bound invaginations of the nuclear envelope proposed to exist initially by Lui et al. (31) and later described by Fricker et al. (15). Because the nuclear envelope and the sarcoplasmic reticulum are continuous, InsP3R activation was expected to lead to calcium release in association with the nuclear region. Our work delineates a role for type 2 InsP3Rs through localization, activation, and gene expression distinct from the role type 2 InsP3Rs may have at the level of the sarcomeres. The separate functions provide a mechanism and support for the hypothesis that calcium concentration fluctuations occur within microdomains in single cells. These regional microdomains explain how an entity, e.g., calcium, can independently control numerous events in a single cell such as growth, development, and contraction.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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