ARTICLE |
Correspondence to: Terje H. Larsen, Experimental Cardiology Unit, Dept. of Radiology, U. of Bergen, Årstadveien 19, N-5009 Bergen, Norway.
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Summary |
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Cardiac myocytes in culture undergo considerable structural reorganization. The remodeling of the myofibrils and the nonmyofibrillar cytoskeleton that occurs in the spreading cardiac myocytes resembles the cellular features observed in the hypertrophying heart. In this study we examined the distribution of the large 60S ribosomal subunit in freshly isolated cardiac myocytes and during the course of attachment and spreading in culture. Initially, anti-60S immunolabeling was scattered widely throughout the sarcoplasm of the dissociated cardiac myocytes. After attachment to the substrate, the 60S ribosomal subunit attained wide sarcoplasmic localization before a sarcomere-related staining pattern appeared in the spreading cell. Double labeling experiments with -actinin confirmed co-localization of the 60S ribosomal subunit with nascent and mature myofibrils. These findings demonstrate that translocation of the 60S ribosomal subunit coincides with the cytoskeletal reorganization taking place in these cells. Moreover, the close association between the myofibrils indicates a particular role for the ribosomes in maintenance and growth of the contractile apparatus. (J Histochem Cytochem 46:963969, 1998)
Key Words: cardiac myocytes, ribosomes, 60S ribosomal subunit, immunofluorescence, microscopy, immunogold electron, microscopy, silver enhancement, myofibrillogenesis, hypertrophy
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Introduction |
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Ribosomes ARE composed of a small 40S and a large 60S ribosomal subunit that form a complex. This ribosomal complex binds mRNA and tRNA and catalyzes peptide bond formation. Frequently the ribosomes occur in clusters, referred to as polyribosomes. Polyribosomes are present in various subcellular compartments as either membrane-bound, cytoskeletal-bound, or as free cytosolic polyribosomes. Whereas the membrane-bound polyribosomes are associated with synthesis of membrane proteins and secretory proteins, the majority of the mRNAs are translated on the other polyribosome fractions. Stimulation of protein synthesis may cause redistribution among the different populations of ribosomes (
In striated muscle cells the myofibrillar components constitute the bulk proportion of proteins. In heart, the half-life of mixed muscle proteins is approximately 10 days (
Isolated cardiac myocytes from neonatal rats rapidly attain a spherical shape in suspension. In culture, these cells attach to the substrate and spread as they undergo considerable structural changes. Considerable reorganization of the normal cellular architecture, including myofibrillar disassembly and reassembly, is apparent within the first hours and days of incubation. It is generally believed that the premyofibril becomes a mature myofibril via a nascent stage by incorporation of certain muscle-specific proteins (
In this study we examined the localization of the 60S ribosomal subunit in freshly isolated and cultured cardiac myocytes, using immunofluorescence staining techniques and immunogold electron microscopy. Redistribution of the 60S ribosomal subunit was demonstrated, along with the considerable structural reorganization of the cells during attachment and spreading.
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Materials and Methods |
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Isolation and Culture of Cardiac Myocytes
Hearts from neonatal rats were washed in Ca++-free Joklik- modified minimal essential medium (MEM) (Gibco Life Technologies; Paisley, UK) with 26.7 mM NaHCO3, 1.20 mM MgSO4, and 1.00 mM DL-carnitine, pH 7.40, and gassed with 5% CO2 in air. After removal of the atria, the ventricular tissue was cut into small pieces and immersed in Ca++-free Joklik-modified MEM containing 0.1% (w/v) collagenase (Worthington Biochemicals; Freehold, NJ) and 0.08% (w/v) bovine serum albumin (BSA) (Sigma Chemical; St Louis, MO). The ventricular tissue was incubated for 60 min in a water bath shaker at 37C and continuously oxygenated. During incubation the enzyme solution was replaced with an identical fresh solution after 30 min. To increase the yield of dissociated myocytes, intact tissue was gently pipetted every 15 min. The cell suspension was filtered through a 200-µm-mesh nylon gauze, washed in Joklik-modified MEM with 1.50 mM Ca++, and the isolated cells collected by centrifugation. The isolated ventricular cells were transferred to a dish containing DMEM (Gibco), 10% fetal calf serum (Gibco), and antibiotics (penicillin 100 IU/ml medium and streptomycin 100 µg/ml medium) (Gibco). After 4 hr of preplating, a large proportion of the nonmyocytes were attached to the dish. The unattached ventricular cells were collected and seeded at a density of 13 x 105 cells/ml in tissue culture dishes (Falcon; Becton Dickinson, Oxford, UK) with or without coverglasses. The cultures were maintained in an atmosphere of 95% air and 5% CO2 at 37C.
Primary Antibodies
Polyclonal rabbit antibodies raised against 60S ribosomal subunits, prepared from rat liver as previously described (-actinin were purchased from Amersham (Poole, UK).
Western Blotting
The protein samples were separated by SDS-PAGE electrophoresis on 10% gels. Protein fractions were then electrophoretically transferred onto a nitrocellulose sheet. Nonspecific binding sites were blocked with 5% BSA (w/v) in 0.1 M PBS containing 0.1% Tween 20 (v/v) before incubation with anti-60S antibodies (diluted 1:400 with 0.8% BSA in PBS/Tween) overnight at 4C. After washing in PBS/Tween, the sheet was incubated with peroxidase-conjugated anti-rabbit antibodies (Dakopatts; Copenhagen, Denmark). The antigenantibodyperoxidase complex was visualized using the ECL chemiluminescence detection kit (Amersham). The specificity of the primary antibodies is shown in Figure 1.
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Immunofluorescence Microscopy
For immunofluorescence microscopy, cardiac cells grown on coverglasses were fixed with 3.7% formaldehyde in 0.1 M PBS after various periods of incubation. In addition, suspensions of freshly isolated cells were fixed and cytospun onto poly-L-lysine-coated coverglasses. Then the cells were permeabilized with 0.5% Triton X-100, treated with 0.2 M glycine, and preincubated with 5% normal swine serum (Dakopatts) and 0.8% BSA (w/v). Incubation with anti-60S antibodies [diluted 1:200 with 0.8% BSA (w/v) in 0.1 M PBS] was performed overnight at 4C. After being rinsed with BSA/PBS, the sections were incubated with TRITC-conjugated swine anti-rabbit antibodies (Dakopatts) at 20C for 1 hr. For double labeling experiments, the cells were further incubated with monoclonal anti--actinin antibodies (diluted 1:200) and then with FITC-conjugated anti-mouse antibodies (Boehringer Mannheim; Mannheim; Germany). Finally, the sections were rinsed in 0.1 M PBS and mounted in a fluorechrome anti-fading solution (H1000; Vector Laboratories, Burlingame, CA) before examination with a fluorescence microscope (Leitz Aristoplane; Leica, Wetzlar, Germany) or a confocal laser scanning microscope (MRC-600; Bio-Rad Microscience, Hertfordshire, UK). The specificity of the immunostaining was controlled by replacing the primary antibodies with normal rabbit serum or a BSA/PBS solution.
Pre-embedding Immunogold Electron Microscopy
Cultured cardiac cells were fixed in situ in the culture dish with 3.7% formaldehyde and 0.1% glutaraldehyde in Hanks' balanced salt solution (Sigma) for 1 hr at room temperature. The cells were permeabilized with 0.1% saponin for 10 min, and 0.01% saponin was used in the buffers during the incubation steps with antibodies. They were treated with glycine, preincubated with normal swine serum, and incubated with primary anti-60S antibodies using a similar protocol to that employed for immunofluorescence staining. The cells were then exposed to secondary antibodies conjugated to ultrasmall gold probes (Aurion; Wageningen, The Netherlands), postfixed with 2% glutaraldehyde, and exposed to a silver enhancement solution as previously described in detail (
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Results |
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Confocal Laser Scanning Microscopy
After seeding, the cardiac cells became rapidly attached to the substrate and started to spread. Initially, the proportion of cardiac myocytes was 8090%, whereas after 6 days in culture the proportion of noncardiac myocytes considerably increased. Prominent sarcoplasmic anti-60S immunostaining was revealed, and in some cardiac myocytes positive labeling was also observed in nucleoli (Figure 2).
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Double labeling of freshly isolated cardiac cells with anti-60S and anti--actinin antibodies showed that only a few cells retained a rod shape (Figure 3A), whereas the majority of cardiac myocytes were spherical (Figure 3B and Figure 3C). Concomitant with spherical transformation, the sarcomeric organization of myofibrils disappeared, as demonstrated by immunolabeling with anti-
-actinin antibodies. During this apparently rapid disassembly of the myofibrils, immunoreactive
-actinin was redistributed to the peripherial proportion of the sarcoplasm. Immunostaining with the anti-60S antibodies revealed specific labeling in the intermyofibrillar and subsarcolemmal areas in the rod-shaped cardiac myocytes. However, in the spherical cells the staining attained a wide distribution in the sarcoplasm without any detectable organization (Figure 3B and Figure 3C). After attachment to the substrate (4 and 8 hr), the sarcoplasmic 60S immunostaining attained a granular appearance (Figure 3DF). At the sites of membrane attachment,
-actinin was accumulated, whereas little 60S immunoreactivity was evident in these areas as revealed by double immunolabeling.
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During further stages of culturing (1224 hr), sarcoplasmic lamellae emerged concomitantly with the appearance of a centrally occurring sarcomeric-like -actinin staining pattern (Figure 3GI). Moreover, strands of
-actinin immunostaining, extending from the centrally occurring myofibrils towards the cell surface, were co-localized with labeling of the 60S ribosomal subunit (Figure 3H and Figure 3I). At later stages of spreading (26 days), the numbers of mature myofibrils increased, along with a redistribution of the 60S ribosomal subunit. In these cardiac myocytes, 60S labeling was present in the intermyofibrillar sarcoplasm (Figure 3J and Figure 3O). Moreover, in some instances an apparent sarcomere-associated immunostaining of the 60S ribosomal subunit occurred in cardiac myocytes containing mature myofibrils (Figure 3N).
Electron Microscopy
The sarcoplasm of the spreading cardiac myocytes contained an abundance of filamentous proteins and mitochondria (Figure 4A). Filaments attached to the substrate were arranged in bundles forming myofibril-like structures (Figure 4B). Silver-enhanced ultrasmall gold particles labeling the 60S ribosomal subunit were seen widely in the sarcoplasm. The specific labeling was frequently closely associated with filaments (Figure 4C) and developing myofibrils (Figure 4B and Figure 5).
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Discussion |
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The dramatic rearrangement of the cell architecture that occurs after isolation and attachment of the cardiac myocytes to the substrate is associated with major changes in the cytoskeleton (-actinin containing dense bodies forms irregular Z-lines to which the thin actin filaments are anchored. Subsequently, muscle myosin II thick filaments as well as other sarcomeric proteins are incorporated into the preformed I-Z-I structures during maturation of the myofibrils (
A close relation of 60S ribosomal subunit staining along the myofibrils was demonstrated in cells co-labeled with anti--actinin antibodies. The 60S ribosomal subunit appeared to be accumulated towards the Z-lines, which is consistent with the ribosome distribution observed in adult skeletal muscle by conventional electron microscopy (
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Acknowledgments |
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Supported by grants from the Norwegian Research Council and the Bergen Heart Foundation.
The skillful technical assistance of Edel Karin Frotjold and Kjellfrid Haukanes is gratefully acknowledged.
Received for publication September 3, 1997; accepted March 11, 1998.
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