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Changes in myofibrils and cytoskeleton of neonatal hamster myocardial cells in culture: An immunofluorescence study
Tissue and Cell 37 (2005) 435–445
Changes in myofibrils and cytoskeleton of neonatal hamster myocardial cells in culture: An immunofluorescence study C. Zhang a , H.E. Osinska b , S.L. Lemanski a , X.P. Huang a , L.F. Lemanski a,∗ a
Department of Biomedical Science, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431, USA b Department of Pediatrics, University of Cincinnati, Cincinnati, OH 45229, USA Received 1 April 2005; received in revised form 9 May 2005; accepted 20 June 2005 Available online 13 September 2005
Abstract Myocardial cells in culture offer many possibilities for studying cellular and molecular biology of cardiac muscles. However, it is important to know how long these cells can be maintained in vitro without significant structural and biochemical changes. In this study, we have investigated the morphological changes of myofibril proteins and cytoskeletons by using immunofluorescent techniques in cultured neonatal hamster myocardial cells at different culture durations. Our results have demonstrated that these cultured cells still contain intact myofibrils and cytoskeletal proteins after 6 days in vitro incubation, however, the organization of some of these proteins is altered. The proteins most sensitive to these in vitro conditions are: myosin heavy chain, actin and desmin. The data indicate that the duration of the culture and the contractile activity of the myocardial cells in culture can influence organization of their contractile apparatus and cytoskeleton. © 2005 Elsevier Ltd. All rights reserved. Keywords: Myofibrils; Myocardial cell; Immunofluorescent microscopy; Hamster
1. Introduction Myocardial cell culture provides us with a practical tool to study various aspects of the biology of cardiac muscle cells, from differentiation to function in normal or pathological states. In order to make the in vitro system a useful model for studying the biology of cardiac myocytes, it is necessary to gain an extensive knowledge of their structural and functional features. Chicken myocardial cells have been used to study myofibrillogenesis (Dlugosz et al., 1984; Sanger et al., 1984, 1986; Lin et al., 1989; Schultheiss et al., 1990; Lu et al., 1992; Wang et al., 1998, 2000). However, it is well known that the avian system has a number of basic differences from mammalian cells and that it is not as well suited as mammals to extrapolate to human heart; for example, differences in the expression of different protein isoforms, lack of T-tubules in avian heart (Manasek, 1968; Jewett et al., 1971) or lack of M-band compartmentation of M-type creatine kinase in avian ∗
Corresponding author. Tel.: +1 561 297 0475; fax: +1 561 297 0422. E-mail address: [email protected] (L.F. Lemanski).
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heart (Wallimann and Eppenberger, 1985). Such observations imply the existence of significant physiological differences between avian and mammalian cardiomyocytes. Several studies have been carried out using cultured mammalian heart cells to investigate the ultrastructure and biochemical changes in long-term culture. However, most of them are mainly focused on the phenomenon of hypertrophy or growth factor expression in cultured myocardial cells (McDermott and Morgan, 1989; Komuro et al., 1991; Donath et al., 1994; Harder et al., 1998). Using living adult cardiomyocytes in culture, Imanaka-Yoshida et al. (1996) observed the fluorescently labeled vinculin and alpha-actinin molecules and costameric structures in these cells. However, only a few immunofluorescent studies have been performed on cultured neonatal mammalian myocardial cells to visualize myofibrils and cytoskeletal organizations. In our laboratory, we have noticed that the cultured mammalian heart cells undergo dedifferentiation or atrophy, and gradually lose their characteristic “muscle-type” morphological characteristics after several days in culture. Interestingly, results of the studies on the role of contraction in the integrity
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of the contractile system in neonatal rat cardiomyocytes in vitro suggest that contractile activity modulates myosin content in these cells (McDermott and Morgan, 1989; Samarel and Engelmann, 1991; Samarel et al., 1992; Qi et al., 1997) as well as their growth and structure (Marino et al., 1987). In the present study, we have investigated the morphological changes of myofibril proteins and cytoskeletons (vinculin, desmoplakin I and II, desmin, titin, myosin, and actin) by using immunofluorescent techniques in cultured neonatal hamster myocardial cells at different culture durations. Our results have demonstrated that these cultured cells still contain intact myofibrils and cytoskeletal proteins after 6 days in vitro incubation, however, the organization of some of these proteins is altered. The proteins most sensitive to these in vitro conditions are: myosin heavy chain, actin and desmin. The data indicate that the duration of the culture and the contractile activity of the myocardial cells in culture can influence organization of their contractile apparatus and cytoskeleton.
2. Materials and methods 2.1. Cell culture Cardiac muscle cells were isolated from the heart ventricles of newborn Syrian hamsters, 2–4 days old, and cultured according to the protocol we described previously (Osinska and Lemanski, 1993). The differential adhesion step was omitted since many cardiomyocytes adhered after only 15 min. The cells were plated at a density of 2.5–3.0 × 105 cells/ml on two-chambered glass slides (0.8 ml of cell suspension per chamber) (Lab-Tek, Detroit, MI, USA) or glass coverslips placed in 35 mm petri dishes (5 ml of cell suspension per dish). Both chamber slides and coverslips were coated with l,l-polylysine (m.wt. 500K) (5 g/ml) and then Vitrogen I (Collagen Corporation, Palo Alto, CA, USA) or Collagen I (Sigma, St. Louis, MO, USA). In order to inhibit proliferation of fibroblasts after 24 h in vitro either glutamine was withdrawn from the culture medium or 100 M cytosine beta-d-arabinofuranoside (Sigma) was added to the culture medium. After 3 or 6 days, the cultured cells were processed for immunofluorescent microscopy. 2.2. Antibodies Several monoclonal antibodies against cytoskeletal proteins were used: (1) desmoplakin I and II (1:10); (2) vinculin
(1:10); (3) titin (1:50); all of these antibodies were purchased from Boehringer-Mannheim (Indianapolis, IN); (4) myosin (MF20 antibody binding to LMM of myosin heavy chain, 1:10) was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biology, University of Iowa, Iowa City, IA; and (5) desmin (polyclonal 1:400) was obtained from Dakopatt Inc. (Carpinteria, CA). Anti-mouse or anti-rabbit antiserum conjugated with Texas red or FITC (Amersham, Arlington Heights, IL) were used as secondary antibodies for indirect fluorescence (1:50 dilution). For the visualization of F-actin, phallacidin fluorescently labeled with BODIPY FL (Molecular Probes, Eugene, OR) was applied. 2.3. Immunofluorescent microscopy For immunofluorescent microscopy, the cells were first washed in PBS and then fixed in 3.7% formaldehyde for 10 min and then permeablized with 0.5% Triton X-100 for 5 min at room temperature and treated with 0.08 M lysine in PBS for 20 min. After blocking with 3% non-fat milk in PBS, the cells were incubated with appropriate primary antibodies for 1 h at 37 ◦ C or overnight at 4 ◦ C and with secondary antibodies for 1 h at room temperature. BODIPY FL-phallacidin was applied together with secondary antibody or in a separate step for 1 h at room temperature. The samples were immersed in 50% glycerol containing 1% n-propyl galate and viewed in a Zeiss Universal light microscope equipped with epifluorescent illumination using a mercury light source and with an additional set of narrow band filters for selective observation of FITC and Texas Red. For controls, staining with secondary antibody only was performed.
3. Results 3.1. Cell viability and beating Most of the cardiomyocytes in our cultures were single, i.e. they were not coupled with each other. After 3 days in vitro, some cells (about 10%) were observed to beat in culture chambers or on coverslips. However, the beating cells decreased after 6 days in culture. Thus, in 6-day-old cultures there were mostly quiescent cardiomyocytes with no more than one or two beating cells per chamber or coverslip.
Fig. 1. Immunostaining of myosin heavy chain and F-actin in cultured hamster myocardial cells. Fluorescence of LMM of myosin heavy chain (Texas Red) (a, b, d, f and h) and F-actin (BODIPY) (c, e and g) of cardiac myocytes cultured for 3 days (a) or 6 days (b–h). (a) Myosin heavy chain in regular striated pattern of A-band staining. Narrow H-line (arrowhead) and wider I-Z-I band (thick arrow) do not show staining. The length of the sarcomere is approximately 2 m. (b) Unusual pattern of myosin heavy chain staining of bright narrow bands at the level of Z-bands and weaker staining at A-band level with bright dots. (c) F-actin in the same cell in narrow bright bands and very weak staining in the A-I band. (d) Myosin heavy chain seen in bands of various width and intensity of staining. (e) F-actin in this cell is absent from areas with the brightest myosin heavy chain staining (arrows). (f) Myosin heavy chain in narrow bands (thin arrow) or wide, brighter patches or amorphous network (thick arrow). (g) F-actin in this cell is in normal pattern (thin arrow) but absent from bands corresponding to bright patches of myosin heavy chain staining (thick arrow), or in nonstriated pattern in the area with amorphous myosin heavy chain staining (arrowhead). (h) Random dots and amorphous staining for myosin heavy chain, ×1120. Bar: 10 m.
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3.2. Myosin heavy chain (MHC) and F-actin In cardiomyocytes cultured for 3 days, most of the cells contained well-organized myofibrils with myosin arranged in A-bands and showing a typical spacing of approximately
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2 m (Fig. 1a). After 6 days in culture, some of the cells still maintained an intact myofibrillar structure and a normal myofibrillar localization of MHC. However, a large proportion of the cells showed obvious abnormalities in the distribution of immunofluorescent staining for MHC. Among them
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were cells with typical wide myofibrils, oriented in the long axis of the cell, but with abnormal spacing of the immunofluorescent staining for MHC (Fig. 1b, d, and f). The cell in Fig. 1b seems to have most of the staining concentrated in narrow bands; bright fluorescent spots of staining of MHC are present at the peripheries of the myofibrils. The staining pattern for F-actin in this cell was also abnormally concentrated in narrow bands (Fig. 1c). Fig. 1d shows another type of abnormality in MHC distribution. Besides the narrow bright bands, the cell shows wide bands of MHC-positive staining irregularly distributed along the myofibrils. The staining for F-actin in this cell is almost normal, except for the wide bands corresponding to the accumulations of MHC-positive staining, which appeared to be F-actin negative (Fig. 1e).
The cell illustrated in Fig. 1f has lost its regular spacing for MHC staining, decreasing about half in comparison with the cells cultured for 3 days. However, MHC-positive staining in the form of an amorphous network is still maintained. The actin fluorescent staining in this cell is almost normal with the exception of a few unstained wide bands corresponding to bright MHC-positive spots (Fig. 1g). In other cells, the immunofluorescent staining for myosin was diffuse, seen almost as an amorphous network with random bright dots and traces of striations at the cell peripheries (Fig. 1h). There were also cells in which the intensity of staining for MHC was very low (data not shown). We further overlaid the fluorescent images from Fig. 1 and tried to observe the structured arrangement of MHC and F-actin staining in the
Fig. 2. Overlay images of double immunofluorescent staining of myosin heavy chain and F-actin in myocardial cells cultured for 6 days. Double immunofluorescence staining of myosin heavy chain (Texas Red, red) and F-actin (BODIPY, green) in the same cells from Fig. 1 indicates that 6-day cultured cardiac myocytes have an abnormal arrangement pattern of myosin heavy chain and F-actin stainings. (a) Overlay images of Fig. 1b and c. (b) Overlay images of Fig. 1d and e. Arrow: brightly stained MHC dot associated with myofibrils. Arrowhead: near Z-line staining of F-actin.
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same cells. Our results revealed an abnormal localization of myosin heavy chain (red) showing four bands in one sarcomere (two brightly stained bands and two weaker stained bands). The two brightly stained bands appear to be located near the Z-lines and overlap with F-actin staining (green) (Fig. 2a). Another abnormality we have noticed is that some MHC staining bands existed in the area where F-actin was negatively stained, suggesting an uneven distribution of MHC and F-actin in myofibrils of these cells (Fig. 2b). 3.3. Titin To further explore the problem of myofibril integrity we used anti-titin monoclonal antibody to visualize eventual changes occurring in the distribution of this protein. In spite of the extensive changes in the patterns of staining for myosin heavy chain and for F-actin, the pattern of titin distribution in the cells cultured for 6 days seemed to be unchanged, compared to the cells cultured for 3 days in vitro. Immunofluorescent staining for titin was seen at the level of I-A-I bands and sometimes as a double band (Fig. 3).
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pattern of myofibril array in the ventral portion of the cell. In many cells there were also foci and single plaques of vinculinpositive fluorescence delineating cell margins (Fig. 4). 3.5. Desmoplakin I and II In single cells cultured for 6 days that did not form desmosomes, due to lack of cell contacts, dots of desmoplakin I and II staining seemed to be randomly distributed on the entire distal surface of the cell including cell processes (Fig. 5a). Careful examination revealed that this staining pattern was correlated with the array of the myofibrils, or rather their nonstriated terminals at the cell peripheries, as seen in phase contrast (Fig. 5b). Occasionally, the distribution of desmoplakin I and II were arranged in rib-like patterns in the central portions of the cells (Fig. 5c). The spacing of the bands was similar to the pattern of costameres observed after anti-vinculin antibody staining, although it was associated with the distal surface of the cell. This rib-like pattern of staining was almost totally absent from the cell peripheries which are usually very flat and close to the substrate (Fig. 5c).
3.4. Vinculin 3.6. Desmin In 6-day-old cultures, immunofluorescent staining for vinculin is seen as extensive bands of costameres in the central portion of the cell and in adhesion plaques at the cell peripheries. This striated pattern of staining is consistent with the
Double staining of MHC (FITC) and desmin (Texas Red) of myocardial cells cultured for 3 days was observed (Fig. 6). Myosin heavy chain staining showed that myofibrils were
Fig. 3. Immunofluorescent staining of titin in hamster myocardial cells cultured for 6 days. Regular striated pattern can be seen in the entire cell, ×810. Bar: 10 m.
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Fig. 4. Immunofluorescent staining of vinculin (biotin–streptavidin–Texas Red) in hamster myocardial cells cultured for 6 days. Striated pattern of vinculin staining can be seen in almost entire area of the cell. The streaks at the cell margin staining of the nucleus are due to the nonspecific binding of biotin, ×810. Bar: 10 m.
oriented in the long axis of the myocardial cells (Fig. 6a). A relatively even distribution of a network of desmin filaments was seen throughout the cytosol (Fig. 6b). Stained A bands were wide and well organized (Fig. 6). After 6 days in vitro, desmin showed two different types of distribution patterns within the cells. There was a very small population of cells that contained desmin associated with the Z-lines. These cells had wide, well-organized myofibrils as seen in phase contrast, and their myosin was arranged in a striated pattern. The vast majority of cells, however, contained desmin arranged partially in a network and partially in a striated pattern (Fig. 7a and c). The myofibrils in these cells were oriented either in the long axis (Fig. 7b) or in multiple axes within a given cell (Fig. 7d). The staining of desmin can be observed more concentrated on the edges of the cells after 6-day cultured compared to the 3-day cultured cells (Fig. 7b and e). The changes of desmin staining in the cells cultured for 6 days are parallel with the changes of myosin staining in the cells at the same culture time.
4. Discussion The MF20 monoclonal antibody against myosin heavy chain (MHC) from striated (chicken pectoralis) muscle,
specifically against its light meromyosin (LMM), binds not only to skeletal muscle (Bader et al., 1982) but also to cultured cardiac myocytes. This antibody does not seem to specifically recognize different isoforms of MHC, but rather an epitope common to both skeletal and cardiac MHC. Thus, the results we have obtained using this antibody illustrate changes in the arrangement of MHC without distinguishing its isoforms. These results, however, corroborate the findings of Marino et al. (1987) who suggested that a combination of contraction, attachment to substrate, as well as nonspecific growth promoters present in serum are all essential for maintaining the ultrastructural integrity of cultured neonatal cardiomyocytes and for stimulating their growth in size. Our observations of a decrease in staining intensity and dispersed staining pattern with the MF20 antibody are also consistent with the findings of McDermott et al. (1987) who found that contraction regulates synthesis of MHC in neonatal cardiomyocytes in vitro, and that the level of MHC decreases in pharmacologically arrested cardiomyocytes. Among the suggested possibilities like a post-transcriptional mechanism by which contraction alters myosin synthesis (McDermott et al., 1987; Goldspink et al., 1996; Qi et al., 1997), or a decrease in a rate of MHC synthesis in arrested cells (Samarel and Engelmann, 1991) or else an increased rate of MHC degradation in arrested cells (Samarel et al., 1992; Eble et al., 1999), the latter seems
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Fig. 5. Immunofluorescent staining of desmoplakin I and II (biotin– streptavidin–Texas Red) in single cardiac myocytes cultured for 6 days. A random pattern of fluorescent dots (a) follows the pattern of array of nonstriated terminals of myofibrils seen in phase contrast (b) (arrows). The striated pattern of desmoplakin I and II can be seen throughout the myocardial cell (c). The staining of the nucleus is due to the nonspecific binding of biotin, ×1120. Bar: 10 m.
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Fig. 6. Double immunofluorescent staining of myosin heavy chain (FITC) and desmin (Texas Red) of hamster myocardial cells cultured for 3 days. Myosin heavy chain staining shows that myofibrils are oriented in the long axis of the cells (a). A relatively even distribution of the network of desmin filaments can be seen throughout the cytosol (b). The staining of A bands is wide and well organized, ×1120.
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Fig. 7. Double immunofluorescent staining of desmin (Texas Red) and myosin heavy chain (FTTC) of hamster myocardial cells cultured for 6 days. Desmin distribution shows two patterns: a network of filaments (arrows) or as delicate bands along myofibrils (arrowheads) (b and e). Myosin heavy chain marking myofibrils oriented in the long axis (b) or in multiple axes (e) of cells, indicating an unusual pattern. The length of a sarcomere is approximately 2 m, but A-bands seem to be narrow (white thin arrow). Unstained H-line (white arrowheads) and I-Z-I bands (white thick arrows) are relatively wide, (c) and (f) are the overlay images of the double staining, ×1120.
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to directly explain our immunofluorescent results with single cardiomyocytes in 6-day cultures. In some of the cells the staining results were negative, while in 3-day cultures all cells were MHC positive, with myosin arranged in typical striated patterns. The abnormal staining patterns for MHC were never seen in short term cultures, which supports the possibility that the abnormalities observed represent various stages of the removal of MHC from the myofibrils. The process decreasing myosin content seems to be a general feature of quiescent cardiomyocytes in vitro. It also has been described in cultures of pharmacologically quiescent cardiomyocytes (Decker et al., 1992a, 1992b; Goldspink et al., 1996; Qi et al., 1997; Horackova et al., 2000). The arrangement of another major myofibrillar protein, Factin, also seems to be affected, but to a lesser degree. In many cells that had lost the striated pattern of MHC distribution, Factin was seen in a striated pattern in I- and A-band locations. However, there were also cells in which F-actin was seen as nonstriated cables. Since the latter cells increased in number with the time in culture, it appears that this was an effect of degradation of myofibrils rather than their reorganization, since they were better organized at earlier times in culture. Our results are consistent to the data previously reported by Sharp et al. (1993). Desmin is the cytoskeletal protein that is considered a marker of myofibril integrity and maturation. It is well known that after plating cardiomyocytes into culture, desmin is at first arranged in a filamentous network and with progressing time in culture becomes redistributed into its original Z-line location (Osinska and Lemanski, 1993). It seems, however, that not all cells in culture have their desmin assembled into Z-lines. Our present observations suggest that a striated pattern for desmin is always present in cells with well-organized myofibrils as seen in phase contrast, and with myosin and actin in their normal striated arrangement. However, in a population of cells cultured for longer times, for example, 6day cultures, the changes in desmin arrangement is observed in parallel with the changes in myosin and F-actin in the cells. It is not clear why desmin in these cells did not rearrange to form Z-line associations. Current models indicate that desmin attaches directly to the Z-line through its interaction with the nebulin c-terminal repeats (Bang et al., 2002). Whether nebulin in the myofibrils stays intact during culture is well worth studying. There could also be a factor other than the presence of myofibrillar proteins in their normal location that plays a role in the association of desmin with Z-discs. Cultured dysgenic mouse myotubes, that have no contractile activity and organized myofibrils, lack mature organization of desmin (Tassin et al., 1988). In cardiac muscle cells from embryonic hearts from 10 days in utero hamsters, the myofibrils are thin, but organization of desmin into Zline register is visible, and these hearts are already beating (Osinska and Lemanski, 1989). Perhaps it is possible that beating is an essential stimulus for the alignment of desmin into the Z-line location. Experiments in which we could follow a particular cardiomyocyte from the culture dish to the
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immunofluorescent microscope would help to answer this question. At present we know that only a small portion of the population of cardiomyocytes in our cultures were beating, and that only a small portion of the cells had desmin in their Z-lines. Moreover, in the cardiomyocytes from cultures of higher cell density, where more of the cells were beating, the striated pattern of desmin staining was seen in many cells (Osinska and Lemanski, 1993). Interestingly, titin distribution remained unchanged during the entire culture period suggesting that this protein is not sensitive to factors such as duration of culture, density of cells in culture and contractility of cells. It is obvious that the presence of titin itself in the myofibrils is not adequate to protect the total integrity of the myofibrils under the conditions of culture we have examined. Apparently, the distribution of one of the major cytoskeletal proteins we studied, vinculin, is not affected by dedifferentiation. Vinculin is one of the components of adherens junctions, by which cells make contacts with neighboring cells or with the extracellular matrix. The actin cytoskeleton of animal cells is anchored to the plasma membrane at these sites (reviewed by Rudiger, 1998). In cardiac myocytes, vinculin is also located at the Z-bands and appears to attach the lateral Z-bands of myofibrils to the plasma membranes and T-tubules via cell-surface integrins (Danowski et al., 1992). The apparently unaffected pattern of vinculin staining supports our speculation that the attachment itself is not enough for maintaining integrity of the contractile machinery of the cardiomyocyte. On the other hand, the unaffected pattern of vinculin staining suggests that these cardiomyocytes did not undergo any mechanical or physiological damage. It has been found, for example, that in ischemic myocytes, the staining pattern for vinculin is changed (Steenbergen et al., 1987). Desmoplakin I and II, generated by alternative splicing from the same gene, are the components contributing to the cytoplasmic dense plaque of the desmosome which anchors the intermediate filaments (Burdett, 1998). The random pattern of distribution of desmoplakin I and II (Fig. 2) was similar to that shown by Atherton et al. (1986), although observations correlating desmoplakin I and II arrangements with the arrays of myofibrils in older cultures to our knowledge has not been examined in detail. The fact that desmoplakin I and II and also vinculin arrangements within the cardiomyocyte do not become disorganized with progressing time in culture suggests that these two proteins are not affected in the process of disintegration of the myofibrils occurring under the conditions of culture in our experiments. Whether random distribution of desmoplakin I and II in single cells has any influence on the integrity of the myofibrils has yet to be established. Whether the mechanisms resulting in a decrease of myofibril protein content such as MHC in both neonatal and adult cardiomyocytes are identical remains to be established although the data from Horackova et al. (2000) did show differences using adult guinea pig cardiomyocytes in long-term
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cultures compared to those previously reported from immature, neonatal myocytes. In summary, our results suggest that several factors could influence integrity of the contractile apparatus of myocardial cells in culture. However, the membrane-associated cytoskeleton seems not affected by these factors. During cell culture or performing cell culture-based experiments, factors that should be taken into our consideration include such factors as the duration of the culture, the density of cultured cells and the contractile activity of myocardial cells in culture.
Acknowledgements The authors are grateful to Masako Nakatsugawa for providing technical support for the studies and to Yuanyuan Jia for typing the manuscript. These studies were supported in part by NIH Grants HL-32184 and HL-37702 and a Christine E. Lynn Grant-in-Aid from the American Heart Association Florida Affiliate to LFL.
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Horackova, M., Morash, B., Byczko, Z, 2000. Altered transarcolemmal Ca transport modifies the myofibrillar ultrastructure and protein metabolism in cultured adult ventricular cardiomyocytes. Mol. Cell. Biochem. 204 (1–2), 21–33. Imanaka-Yoshida, K., Danowski, B.A., Sanger, J.M., Sanger, J.W., 1996. Living adult rat cardiomyocytes in culture: evidence for dissociation of costameric distribution of vinculin from costameric distributions of attachments. Cell Motil. Cytoskeleton 33 (4), 263–275. Jewett, P.H., Sommer, J.R., Johnson, E.A., 1971. Cardiac muscle. Its ultrastructure in the finch and hummingbird with special reference to the sarcoplasmic reticulum. J. Cell. Biol. 49, 50–65. Komuro, I., Katoh, Y., Kaida, T., Shibazaki, Y., Kurabayashi, M., Hoh, E., Takau, F., Yazaki, Y., 1991. Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. J. Biol. Chem. 266, 1265–1268. Lin, Z., Holtzer, S., Schulthesis, T., Murray, J., Masaki, T., Fischman, D.A., Holtzer, H., 1989. Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes. J. Cell. Biol. 108, 2355–2367. Lu, M.-H., DiLullo, C., Schultheiss, T., Holtzer, S., Murray, J.M., Choi, J., Fischman, D.A., Holtzer, H., 1992. The vinculin/sarcomeric-alphaactinin/alpha-actin nexus in cultured cardiac myocytes. J. Cell. Biol. 117, 1007–1022. Marino, T.A., Kuseryk, L., Lauva, I.K., 1987. Role of contrction in the structures and growth of neonatal rat cardiocytes. Am. J. Physiol. 253, H1391–H1399. Manasek, F.J., 1968. Embryonic development of the heart. I. A light anf electron microscopic study of myocardial development in the early chick embryo. J. Morphol. 125, 329–366. McDermott, P.J., Whitaker-Dowling, P., Klein, I., 1987. Regulation of cardiac myosin synthesis: studies of RNA content in cultured heart cells. Exp. Cell Res. 173, 183–192. McDermott, P.J., Morgan, H.E., 1989. Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture. Circ. Res. 64, 542–553. Osinska, H.E., Lemanski, L.F., 1989. Immunofluorescent localization of desmin and vimentin in developing cardiac muscle of Syrian hamster. Anat. Rec. 223 (4), 406–413. Osinska, H.E., Lemanski, L.F., 1993. Immunofluorescent studies on Zline-associated protein in cultured cardiomyocytes from neonatal hamsters. Cell Tissue Res. 271, 59–67. Qi, M., Puglisi, J.L., Byron, K.L., Ojamaa, K., Klein, L., Bers, D.M., Samarel, A.M., 1997. Myosin heavy chain gene expression in neonatal rat heart cells: effects of [Ca2+ ]i and contractile activity. Am. J. Physiol. 273 (2 Pt 1), C394–C403. Rudiger, M., 1998. Vinculin and alpha-catenin: shared and unique functions in adherens junctions. Bioessays 20 (9), 733–740. Samarel, A.M., Engelmann, G.L., 1991. Contractile activity modulates myosin heavy chain-beta expression in neonatal rat heart cells. Am. J. Physiol. 261, H1067–H1077. Samarel, A.M., Spragia, M.L., Maloney, V., Kamal, S.A., Engelmann, G.L., 1992. Contractile arrest accelerates myosin heavy chain degradation in neonatal rat heart cells. Am. J. Physiol. 263, C642–C652. Sanger, J.W., Mittal, B., Sanger, J.M., 1984. Formation of myofibrils in spreading chick cardiac myocytes. Cell Motil. 4, 405–416. Sanger, J.M., Mittal, B., Pochapin, M.B., Sanger, J.W., 1986. Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin. J. Cell Biol. 102, 2053–2066. Schultheiss, T., Lin, Z., Lu, M.-H., Murray, J., Fischman, D.A., Weber, K., Masaki, T., Imamura, M., Holtzer, H., 1990. Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils. J. Cell Biol. 110, 1159–1172. Sharp, W.W., Terracio, L., Borg, T.K., Samarel, A.M., 1993. Contractile activity modulates actin synthesis and turnover in cultured neonatal rat heart cells. Circ. Res. 73 (1), 172–183. Steenbergen, C., Hill, M.L., Jennings, R.B., 1987. Cytoskeletal damage during myocardial ischemia: changes in vinculin immunofluorescence
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Changes in myofibrils and cytoskeleton of neonatal hamster myocardial cells in culture: An immunofluorescence study C. Zhang a , H.E. Osinska b , S.L. Lemanski a , X.P. Huang a , L.F. Lemanski a,∗ a
Department of Biomedical Science, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431, USA b Department of Pediatrics, University of Cincinnati, Cincinnati, OH 45229, USA Received 1 April 2005; received in revised form 9 May 2005; accepted 20 June 2005 Available online 13 September 2005
Abstract Myocardial cells in culture offer many possibilities for studying cellular and molecular biology of cardiac muscles. However, it is important to know how long these cells can be maintained in vitro without significant structural and biochemical changes. In this study, we have investigated the morphological changes of myofibril proteins and cytoskeletons by using immunofluorescent techniques in cultured neonatal hamster myocardial cells at different culture durations. Our results have demonstrated that these cultured cells still contain intact myofibrils and cytoskeletal proteins after 6 days in vitro incubation, however, the organization of some of these proteins is altered. The proteins most sensitive to these in vitro conditions are: myosin heavy chain, actin and desmin. The data indicate that the duration of the culture and the contractile activity of the myocardial cells in culture can influence organization of their contractile apparatus and cytoskeleton. © 2005 Elsevier Ltd. All rights reserved. Keywords: Myofibrils; Myocardial cell; Immunofluorescent microscopy; Hamster
1. Introduction Myocardial cell culture provides us with a practical tool to study various aspects of the biology of cardiac muscle cells, from differentiation to function in normal or pathological states. In order to make the in vitro system a useful model for studying the biology of cardiac myocytes, it is necessary to gain an extensive knowledge of their structural and functional features. Chicken myocardial cells have been used to study myofibrillogenesis (Dlugosz et al., 1984; Sanger et al., 1984, 1986; Lin et al., 1989; Schultheiss et al., 1990; Lu et al., 1992; Wang et al., 1998, 2000). However, it is well known that the avian system has a number of basic differences from mammalian cells and that it is not as well suited as mammals to extrapolate to human heart; for example, differences in the expression of different protein isoforms, lack of T-tubules in avian heart (Manasek, 1968; Jewett et al., 1971) or lack of M-band compartmentation of M-type creatine kinase in avian ∗
Corresponding author. Tel.: +1 561 297 0475; fax: +1 561 297 0422. E-mail address: [email protected] (L.F. Lemanski).
0040-8166/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2005.06.004
heart (Wallimann and Eppenberger, 1985). Such observations imply the existence of significant physiological differences between avian and mammalian cardiomyocytes. Several studies have been carried out using cultured mammalian heart cells to investigate the ultrastructure and biochemical changes in long-term culture. However, most of them are mainly focused on the phenomenon of hypertrophy or growth factor expression in cultured myocardial cells (McDermott and Morgan, 1989; Komuro et al., 1991; Donath et al., 1994; Harder et al., 1998). Using living adult cardiomyocytes in culture, Imanaka-Yoshida et al. (1996) observed the fluorescently labeled vinculin and alpha-actinin molecules and costameric structures in these cells. However, only a few immunofluorescent studies have been performed on cultured neonatal mammalian myocardial cells to visualize myofibrils and cytoskeletal organizations. In our laboratory, we have noticed that the cultured mammalian heart cells undergo dedifferentiation or atrophy, and gradually lose their characteristic “muscle-type” morphological characteristics after several days in culture. Interestingly, results of the studies on the role of contraction in the integrity
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of the contractile system in neonatal rat cardiomyocytes in vitro suggest that contractile activity modulates myosin content in these cells (McDermott and Morgan, 1989; Samarel and Engelmann, 1991; Samarel et al., 1992; Qi et al., 1997) as well as their growth and structure (Marino et al., 1987). In the present study, we have investigated the morphological changes of myofibril proteins and cytoskeletons (vinculin, desmoplakin I and II, desmin, titin, myosin, and actin) by using immunofluorescent techniques in cultured neonatal hamster myocardial cells at different culture durations. Our results have demonstrated that these cultured cells still contain intact myofibrils and cytoskeletal proteins after 6 days in vitro incubation, however, the organization of some of these proteins is altered. The proteins most sensitive to these in vitro conditions are: myosin heavy chain, actin and desmin. The data indicate that the duration of the culture and the contractile activity of the myocardial cells in culture can influence organization of their contractile apparatus and cytoskeleton.
2. Materials and methods 2.1. Cell culture Cardiac muscle cells were isolated from the heart ventricles of newborn Syrian hamsters, 2–4 days old, and cultured according to the protocol we described previously (Osinska and Lemanski, 1993). The differential adhesion step was omitted since many cardiomyocytes adhered after only 15 min. The cells were plated at a density of 2.5–3.0 × 105 cells/ml on two-chambered glass slides (0.8 ml of cell suspension per chamber) (Lab-Tek, Detroit, MI, USA) or glass coverslips placed in 35 mm petri dishes (5 ml of cell suspension per dish). Both chamber slides and coverslips were coated with l,l-polylysine (m.wt. 500K) (5 g/ml) and then Vitrogen I (Collagen Corporation, Palo Alto, CA, USA) or Collagen I (Sigma, St. Louis, MO, USA). In order to inhibit proliferation of fibroblasts after 24 h in vitro either glutamine was withdrawn from the culture medium or 100 M cytosine beta-d-arabinofuranoside (Sigma) was added to the culture medium. After 3 or 6 days, the cultured cells were processed for immunofluorescent microscopy. 2.2. Antibodies Several monoclonal antibodies against cytoskeletal proteins were used: (1) desmoplakin I and II (1:10); (2) vinculin
(1:10); (3) titin (1:50); all of these antibodies were purchased from Boehringer-Mannheim (Indianapolis, IN); (4) myosin (MF20 antibody binding to LMM of myosin heavy chain, 1:10) was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biology, University of Iowa, Iowa City, IA; and (5) desmin (polyclonal 1:400) was obtained from Dakopatt Inc. (Carpinteria, CA). Anti-mouse or anti-rabbit antiserum conjugated with Texas red or FITC (Amersham, Arlington Heights, IL) were used as secondary antibodies for indirect fluorescence (1:50 dilution). For the visualization of F-actin, phallacidin fluorescently labeled with BODIPY FL (Molecular Probes, Eugene, OR) was applied. 2.3. Immunofluorescent microscopy For immunofluorescent microscopy, the cells were first washed in PBS and then fixed in 3.7% formaldehyde for 10 min and then permeablized with 0.5% Triton X-100 for 5 min at room temperature and treated with 0.08 M lysine in PBS for 20 min. After blocking with 3% non-fat milk in PBS, the cells were incubated with appropriate primary antibodies for 1 h at 37 ◦ C or overnight at 4 ◦ C and with secondary antibodies for 1 h at room temperature. BODIPY FL-phallacidin was applied together with secondary antibody or in a separate step for 1 h at room temperature. The samples were immersed in 50% glycerol containing 1% n-propyl galate and viewed in a Zeiss Universal light microscope equipped with epifluorescent illumination using a mercury light source and with an additional set of narrow band filters for selective observation of FITC and Texas Red. For controls, staining with secondary antibody only was performed.
3. Results 3.1. Cell viability and beating Most of the cardiomyocytes in our cultures were single, i.e. they were not coupled with each other. After 3 days in vitro, some cells (about 10%) were observed to beat in culture chambers or on coverslips. However, the beating cells decreased after 6 days in culture. Thus, in 6-day-old cultures there were mostly quiescent cardiomyocytes with no more than one or two beating cells per chamber or coverslip.
Fig. 1. Immunostaining of myosin heavy chain and F-actin in cultured hamster myocardial cells. Fluorescence of LMM of myosin heavy chain (Texas Red) (a, b, d, f and h) and F-actin (BODIPY) (c, e and g) of cardiac myocytes cultured for 3 days (a) or 6 days (b–h). (a) Myosin heavy chain in regular striated pattern of A-band staining. Narrow H-line (arrowhead) and wider I-Z-I band (thick arrow) do not show staining. The length of the sarcomere is approximately 2 m. (b) Unusual pattern of myosin heavy chain staining of bright narrow bands at the level of Z-bands and weaker staining at A-band level with bright dots. (c) F-actin in the same cell in narrow bright bands and very weak staining in the A-I band. (d) Myosin heavy chain seen in bands of various width and intensity of staining. (e) F-actin in this cell is absent from areas with the brightest myosin heavy chain staining (arrows). (f) Myosin heavy chain in narrow bands (thin arrow) or wide, brighter patches or amorphous network (thick arrow). (g) F-actin in this cell is in normal pattern (thin arrow) but absent from bands corresponding to bright patches of myosin heavy chain staining (thick arrow), or in nonstriated pattern in the area with amorphous myosin heavy chain staining (arrowhead). (h) Random dots and amorphous staining for myosin heavy chain, ×1120. Bar: 10 m.
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3.2. Myosin heavy chain (MHC) and F-actin In cardiomyocytes cultured for 3 days, most of the cells contained well-organized myofibrils with myosin arranged in A-bands and showing a typical spacing of approximately
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2 m (Fig. 1a). After 6 days in culture, some of the cells still maintained an intact myofibrillar structure and a normal myofibrillar localization of MHC. However, a large proportion of the cells showed obvious abnormalities in the distribution of immunofluorescent staining for MHC. Among them
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were cells with typical wide myofibrils, oriented in the long axis of the cell, but with abnormal spacing of the immunofluorescent staining for MHC (Fig. 1b, d, and f). The cell in Fig. 1b seems to have most of the staining concentrated in narrow bands; bright fluorescent spots of staining of MHC are present at the peripheries of the myofibrils. The staining pattern for F-actin in this cell was also abnormally concentrated in narrow bands (Fig. 1c). Fig. 1d shows another type of abnormality in MHC distribution. Besides the narrow bright bands, the cell shows wide bands of MHC-positive staining irregularly distributed along the myofibrils. The staining for F-actin in this cell is almost normal, except for the wide bands corresponding to the accumulations of MHC-positive staining, which appeared to be F-actin negative (Fig. 1e).
The cell illustrated in Fig. 1f has lost its regular spacing for MHC staining, decreasing about half in comparison with the cells cultured for 3 days. However, MHC-positive staining in the form of an amorphous network is still maintained. The actin fluorescent staining in this cell is almost normal with the exception of a few unstained wide bands corresponding to bright MHC-positive spots (Fig. 1g). In other cells, the immunofluorescent staining for myosin was diffuse, seen almost as an amorphous network with random bright dots and traces of striations at the cell peripheries (Fig. 1h). There were also cells in which the intensity of staining for MHC was very low (data not shown). We further overlaid the fluorescent images from Fig. 1 and tried to observe the structured arrangement of MHC and F-actin staining in the
Fig. 2. Overlay images of double immunofluorescent staining of myosin heavy chain and F-actin in myocardial cells cultured for 6 days. Double immunofluorescence staining of myosin heavy chain (Texas Red, red) and F-actin (BODIPY, green) in the same cells from Fig. 1 indicates that 6-day cultured cardiac myocytes have an abnormal arrangement pattern of myosin heavy chain and F-actin stainings. (a) Overlay images of Fig. 1b and c. (b) Overlay images of Fig. 1d and e. Arrow: brightly stained MHC dot associated with myofibrils. Arrowhead: near Z-line staining of F-actin.
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same cells. Our results revealed an abnormal localization of myosin heavy chain (red) showing four bands in one sarcomere (two brightly stained bands and two weaker stained bands). The two brightly stained bands appear to be located near the Z-lines and overlap with F-actin staining (green) (Fig. 2a). Another abnormality we have noticed is that some MHC staining bands existed in the area where F-actin was negatively stained, suggesting an uneven distribution of MHC and F-actin in myofibrils of these cells (Fig. 2b). 3.3. Titin To further explore the problem of myofibril integrity we used anti-titin monoclonal antibody to visualize eventual changes occurring in the distribution of this protein. In spite of the extensive changes in the patterns of staining for myosin heavy chain and for F-actin, the pattern of titin distribution in the cells cultured for 6 days seemed to be unchanged, compared to the cells cultured for 3 days in vitro. Immunofluorescent staining for titin was seen at the level of I-A-I bands and sometimes as a double band (Fig. 3).
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pattern of myofibril array in the ventral portion of the cell. In many cells there were also foci and single plaques of vinculinpositive fluorescence delineating cell margins (Fig. 4). 3.5. Desmoplakin I and II In single cells cultured for 6 days that did not form desmosomes, due to lack of cell contacts, dots of desmoplakin I and II staining seemed to be randomly distributed on the entire distal surface of the cell including cell processes (Fig. 5a). Careful examination revealed that this staining pattern was correlated with the array of the myofibrils, or rather their nonstriated terminals at the cell peripheries, as seen in phase contrast (Fig. 5b). Occasionally, the distribution of desmoplakin I and II were arranged in rib-like patterns in the central portions of the cells (Fig. 5c). The spacing of the bands was similar to the pattern of costameres observed after anti-vinculin antibody staining, although it was associated with the distal surface of the cell. This rib-like pattern of staining was almost totally absent from the cell peripheries which are usually very flat and close to the substrate (Fig. 5c).
3.4. Vinculin 3.6. Desmin In 6-day-old cultures, immunofluorescent staining for vinculin is seen as extensive bands of costameres in the central portion of the cell and in adhesion plaques at the cell peripheries. This striated pattern of staining is consistent with the
Double staining of MHC (FITC) and desmin (Texas Red) of myocardial cells cultured for 3 days was observed (Fig. 6). Myosin heavy chain staining showed that myofibrils were
Fig. 3. Immunofluorescent staining of titin in hamster myocardial cells cultured for 6 days. Regular striated pattern can be seen in the entire cell, ×810. Bar: 10 m.
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Fig. 4. Immunofluorescent staining of vinculin (biotin–streptavidin–Texas Red) in hamster myocardial cells cultured for 6 days. Striated pattern of vinculin staining can be seen in almost entire area of the cell. The streaks at the cell margin staining of the nucleus are due to the nonspecific binding of biotin, ×810. Bar: 10 m.
oriented in the long axis of the myocardial cells (Fig. 6a). A relatively even distribution of a network of desmin filaments was seen throughout the cytosol (Fig. 6b). Stained A bands were wide and well organized (Fig. 6). After 6 days in vitro, desmin showed two different types of distribution patterns within the cells. There was a very small population of cells that contained desmin associated with the Z-lines. These cells had wide, well-organized myofibrils as seen in phase contrast, and their myosin was arranged in a striated pattern. The vast majority of cells, however, contained desmin arranged partially in a network and partially in a striated pattern (Fig. 7a and c). The myofibrils in these cells were oriented either in the long axis (Fig. 7b) or in multiple axes within a given cell (Fig. 7d). The staining of desmin can be observed more concentrated on the edges of the cells after 6-day cultured compared to the 3-day cultured cells (Fig. 7b and e). The changes of desmin staining in the cells cultured for 6 days are parallel with the changes of myosin staining in the cells at the same culture time.
4. Discussion The MF20 monoclonal antibody against myosin heavy chain (MHC) from striated (chicken pectoralis) muscle,
specifically against its light meromyosin (LMM), binds not only to skeletal muscle (Bader et al., 1982) but also to cultured cardiac myocytes. This antibody does not seem to specifically recognize different isoforms of MHC, but rather an epitope common to both skeletal and cardiac MHC. Thus, the results we have obtained using this antibody illustrate changes in the arrangement of MHC without distinguishing its isoforms. These results, however, corroborate the findings of Marino et al. (1987) who suggested that a combination of contraction, attachment to substrate, as well as nonspecific growth promoters present in serum are all essential for maintaining the ultrastructural integrity of cultured neonatal cardiomyocytes and for stimulating their growth in size. Our observations of a decrease in staining intensity and dispersed staining pattern with the MF20 antibody are also consistent with the findings of McDermott et al. (1987) who found that contraction regulates synthesis of MHC in neonatal cardiomyocytes in vitro, and that the level of MHC decreases in pharmacologically arrested cardiomyocytes. Among the suggested possibilities like a post-transcriptional mechanism by which contraction alters myosin synthesis (McDermott et al., 1987; Goldspink et al., 1996; Qi et al., 1997), or a decrease in a rate of MHC synthesis in arrested cells (Samarel and Engelmann, 1991) or else an increased rate of MHC degradation in arrested cells (Samarel et al., 1992; Eble et al., 1999), the latter seems
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Fig. 5. Immunofluorescent staining of desmoplakin I and II (biotin– streptavidin–Texas Red) in single cardiac myocytes cultured for 6 days. A random pattern of fluorescent dots (a) follows the pattern of array of nonstriated terminals of myofibrils seen in phase contrast (b) (arrows). The striated pattern of desmoplakin I and II can be seen throughout the myocardial cell (c). The staining of the nucleus is due to the nonspecific binding of biotin, ×1120. Bar: 10 m.
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Fig. 6. Double immunofluorescent staining of myosin heavy chain (FITC) and desmin (Texas Red) of hamster myocardial cells cultured for 3 days. Myosin heavy chain staining shows that myofibrils are oriented in the long axis of the cells (a). A relatively even distribution of the network of desmin filaments can be seen throughout the cytosol (b). The staining of A bands is wide and well organized, ×1120.
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Fig. 7. Double immunofluorescent staining of desmin (Texas Red) and myosin heavy chain (FTTC) of hamster myocardial cells cultured for 6 days. Desmin distribution shows two patterns: a network of filaments (arrows) or as delicate bands along myofibrils (arrowheads) (b and e). Myosin heavy chain marking myofibrils oriented in the long axis (b) or in multiple axes (e) of cells, indicating an unusual pattern. The length of a sarcomere is approximately 2 m, but A-bands seem to be narrow (white thin arrow). Unstained H-line (white arrowheads) and I-Z-I bands (white thick arrows) are relatively wide, (c) and (f) are the overlay images of the double staining, ×1120.
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to directly explain our immunofluorescent results with single cardiomyocytes in 6-day cultures. In some of the cells the staining results were negative, while in 3-day cultures all cells were MHC positive, with myosin arranged in typical striated patterns. The abnormal staining patterns for MHC were never seen in short term cultures, which supports the possibility that the abnormalities observed represent various stages of the removal of MHC from the myofibrils. The process decreasing myosin content seems to be a general feature of quiescent cardiomyocytes in vitro. It also has been described in cultures of pharmacologically quiescent cardiomyocytes (Decker et al., 1992a, 1992b; Goldspink et al., 1996; Qi et al., 1997; Horackova et al., 2000). The arrangement of another major myofibrillar protein, Factin, also seems to be affected, but to a lesser degree. In many cells that had lost the striated pattern of MHC distribution, Factin was seen in a striated pattern in I- and A-band locations. However, there were also cells in which F-actin was seen as nonstriated cables. Since the latter cells increased in number with the time in culture, it appears that this was an effect of degradation of myofibrils rather than their reorganization, since they were better organized at earlier times in culture. Our results are consistent to the data previously reported by Sharp et al. (1993). Desmin is the cytoskeletal protein that is considered a marker of myofibril integrity and maturation. It is well known that after plating cardiomyocytes into culture, desmin is at first arranged in a filamentous network and with progressing time in culture becomes redistributed into its original Z-line location (Osinska and Lemanski, 1993). It seems, however, that not all cells in culture have their desmin assembled into Z-lines. Our present observations suggest that a striated pattern for desmin is always present in cells with well-organized myofibrils as seen in phase contrast, and with myosin and actin in their normal striated arrangement. However, in a population of cells cultured for longer times, for example, 6day cultures, the changes in desmin arrangement is observed in parallel with the changes in myosin and F-actin in the cells. It is not clear why desmin in these cells did not rearrange to form Z-line associations. Current models indicate that desmin attaches directly to the Z-line through its interaction with the nebulin c-terminal repeats (Bang et al., 2002). Whether nebulin in the myofibrils stays intact during culture is well worth studying. There could also be a factor other than the presence of myofibrillar proteins in their normal location that plays a role in the association of desmin with Z-discs. Cultured dysgenic mouse myotubes, that have no contractile activity and organized myofibrils, lack mature organization of desmin (Tassin et al., 1988). In cardiac muscle cells from embryonic hearts from 10 days in utero hamsters, the myofibrils are thin, but organization of desmin into Zline register is visible, and these hearts are already beating (Osinska and Lemanski, 1989). Perhaps it is possible that beating is an essential stimulus for the alignment of desmin into the Z-line location. Experiments in which we could follow a particular cardiomyocyte from the culture dish to the
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immunofluorescent microscope would help to answer this question. At present we know that only a small portion of the population of cardiomyocytes in our cultures were beating, and that only a small portion of the cells had desmin in their Z-lines. Moreover, in the cardiomyocytes from cultures of higher cell density, where more of the cells were beating, the striated pattern of desmin staining was seen in many cells (Osinska and Lemanski, 1993). Interestingly, titin distribution remained unchanged during the entire culture period suggesting that this protein is not sensitive to factors such as duration of culture, density of cells in culture and contractility of cells. It is obvious that the presence of titin itself in the myofibrils is not adequate to protect the total integrity of the myofibrils under the conditions of culture we have examined. Apparently, the distribution of one of the major cytoskeletal proteins we studied, vinculin, is not affected by dedifferentiation. Vinculin is one of the components of adherens junctions, by which cells make contacts with neighboring cells or with the extracellular matrix. The actin cytoskeleton of animal cells is anchored to the plasma membrane at these sites (reviewed by Rudiger, 1998). In cardiac myocytes, vinculin is also located at the Z-bands and appears to attach the lateral Z-bands of myofibrils to the plasma membranes and T-tubules via cell-surface integrins (Danowski et al., 1992). The apparently unaffected pattern of vinculin staining supports our speculation that the attachment itself is not enough for maintaining integrity of the contractile machinery of the cardiomyocyte. On the other hand, the unaffected pattern of vinculin staining suggests that these cardiomyocytes did not undergo any mechanical or physiological damage. It has been found, for example, that in ischemic myocytes, the staining pattern for vinculin is changed (Steenbergen et al., 1987). Desmoplakin I and II, generated by alternative splicing from the same gene, are the components contributing to the cytoplasmic dense plaque of the desmosome which anchors the intermediate filaments (Burdett, 1998). The random pattern of distribution of desmoplakin I and II (Fig. 2) was similar to that shown by Atherton et al. (1986), although observations correlating desmoplakin I and II arrangements with the arrays of myofibrils in older cultures to our knowledge has not been examined in detail. The fact that desmoplakin I and II and also vinculin arrangements within the cardiomyocyte do not become disorganized with progressing time in culture suggests that these two proteins are not affected in the process of disintegration of the myofibrils occurring under the conditions of culture in our experiments. Whether random distribution of desmoplakin I and II in single cells has any influence on the integrity of the myofibrils has yet to be established. Whether the mechanisms resulting in a decrease of myofibril protein content such as MHC in both neonatal and adult cardiomyocytes are identical remains to be established although the data from Horackova et al. (2000) did show differences using adult guinea pig cardiomyocytes in long-term
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cultures compared to those previously reported from immature, neonatal myocytes. In summary, our results suggest that several factors could influence integrity of the contractile apparatus of myocardial cells in culture. However, the membrane-associated cytoskeleton seems not affected by these factors. During cell culture or performing cell culture-based experiments, factors that should be taken into our consideration include such factors as the duration of the culture, the density of cultured cells and the contractile activity of myocardial cells in culture.
Acknowledgements The authors are grateful to Masako Nakatsugawa for providing technical support for the studies and to Yuanyuan Jia for typing the manuscript. These studies were supported in part by NIH Grants HL-32184 and HL-37702 and a Christine E. Lynn Grant-in-Aid from the American Heart Association Florida Affiliate to LFL.
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