Immunofluorescent studies for ╬▒-actinin in cultured cardiomyopathic hamster heart cells.код для вставкиСкачать
THE ANATOMICAL RECORD 228:46-52 (1990) lmmunofluorescent Studies for cx-Actinin in Cultured Cardiomyopathic Hamster Heart Cells JIAN LI AND LARRY F. LEMANSKI Department of Anatomy and Cell Biology, State University of New York, Health Science Center at Syracuse, Syracuse, New York ABSTRACT Primary cultures of cardiac myocytes from normal and genetically cardiomyopathic (CM) newborn hamsters (strain UM X7.1) were analyzed by indirect immunofluorescent microscopy after 3, 5, 7, and 9 days in culture. The cultures were fixed in cold acetone and immunostained by a n indirect method using FITC-labelled anti-a-actinin to label the myofibrillar Z bands. Most normal and CM myocytes appeared round in shape after 3 days in culture. Normal cardiac myocytes began to exhibit cytoplasmic projections after 5 days in culture and their myofibrils usually showed parallel arrangements with respect to each other. The cardiac cells from CM hearts showed a n obvious myofibril disarray. Moreover, projections formed later than normal. As the size of the cells increased, more and more projections formed in normal hamster myocytes during development. By contrast, most of the cardiomyopathic myocytes showed few projections even as late a s 9 days in culture. Hence, the number of projections per cell was much less in cardiomyopathic myocytes than in normal, especially after 7 and 9 days in culture. These results suggest that cardiomyopathic cells have abnormal shapes in culture and, in particular, fail to form projections as in normal cells. Whether this unusual behavior is related to a n abnormality of the membranes or cytoskeletal system in cardiomyopathic heart cells or to some other factor requires further study. Many cardiomyopathies are known to be inherited conditions t h a t begin in the developing heart before birth. In humans, cardiomyopathies often result in early death, usually before or during puberty (Ferrans et al., 1972; Goodwin, 1985). The etiologies of most cardiomyopathies remain unknown. The cardiomyopathic hamster is a reproducible spontaneous model of chronic congestive heart failure and thus may be a useful paradigm for human myocardial disease. The defect in hamsters purportedly is transmitted by a n autosomal recessive gene (Homburger et al., 1962). Recently, several investigators have shown that this genetic animal model closely mimics certain common human cardiomyopathies, such as asymmetric hypertrophy (ASH) and idiopathic hypertrophic subaortic stenosis (IHSS). For example, the disorientation of myofibrils, a rather classical trait of hypertrophic obstructive cardiomyopathies in humans (Heggtveit and Nadkarni, 1971; Ferrans et al., 1972), also is commonly seen in cardiomyopathic hamster heart cells (Lemanski and Tu, 1983). Moreover, i t has been shown that the average size of myocytes from cardiomyopathic hamster heart is larger than that from control hamsters of similar ages (Sorenson et al., 1985). Other prominent features in CM hamster hearts consist of hypercontraction of cardiocytes, and disruption of intercalated discs (Jasmin and Eu, 1979). In previous work from our laboratory, it was demonstrated that the cardiomyopathic myocardial cells show abnormal morphologies after 3 to 5 days in culture (Lemanski and Tu,1983). An abnormally thick ventricu0 1990 WILEY-LISS. INC lar wall was noted in newborn CM hearts suggesting that the organ is already affected at birth (Lemanski et al., 1990). The present study was undertaken to analyze the morphologies of cardiomyocytes from normal and cardiomyopathic hamsters at different times in vitro. In order to observe the developing myofibrils better, immunofluorescent methods using anti-a-actinin to stain the Z bands were employed. Our results show that cardiomyopathic heart cells have significant myofibril disarray when compared with normal. In addition, the size and number of cellular projections from cardiomyopathic hamster hearts were less than on myocytes of normal hamster beginning at 5 days in culture. This difference became more pronounced with increasing time in culture. MATERIALS A N D METHODS Animals Genetically cardiomyopathic, strain UM-X7.1 and control Syrian hamsters were obtained from our breeding colony maintained in the Central Animal Care Facility a t the State University of New York, Health Science Center a t Syracuse. The normal and CM animals were housed under identical conditions in the same Received October 28, 1989; accepted January 29, 1990. Address reprint requests to Dr. Larry F. Lemanski, Department of Anatomy and Cell Biology, SUNY Health Science Center, 750 East Adams Street, Syracuse, NY 13210. a-ACTININ IN CARDIOMYOPATHIC HAMSTER HEART room a t 23°C on a light cycle of 12 h r light-12 h r dark. They were fed Purina Lab Chow and water ad libitum with a supplement of hamster seed mixture and lettuce. Cell Isolation and Culture Myocytes were isolated from heart ventricles of 3 day old normal and cardiomyopathic hamsters. Between 20 and 50 animals from each strain were used in each experiment. The experiments were carried out a total of 5 times. The animals were killed by cervical dislocation and the hearts immediately removed using sterile techniques. The extirpated hearts were washed 2 times in cold Hanks’ solution to remove residual blood. The ventricles were dissected free and minced into very small pieces by using a new scalpel under a dissecting microscope. The pieces were washed twice with ice cold Hanks’ solution and then treated with 0.08% trypsin and 0.01% collagenase in Hanks’ solution for 10 min a t 37°C in a n incubator with gentle agitation. The first supernatant was discarded and the three additional supernatants were diluted twofold with cold culture medium containing Earle’s minimum essential medium, 15% fetal calf serum, 200 mM glutamine, 100 u/ml penicillin, and 100 mg/ml streptomycin. Cells were harvested from the enzymatic solution by centrifugation, and fresh culture medium was added to resuspend the cells. In order to enrich for myocytes, a differential adhesion step was used. To accomplish this, the dissociated heart cell suspensions were preincubated in a culture dish for 60 min at 37°C. Most of the fibroblasts attached to the bottom of the dish during this period. The remaining unattached cells (containing mostly myocytes) were diluted to a n average density of 2 x lo5 dispersed celldm1 of medium. The cells were grown on 22 mm2 gelatin-coated glass microscope coverslips in 35 mm diameter plastic tissue culture dishes a t 37°C in a 5% carbon dioxide and 95% air mixture. The culture medium was changed every other day. For the present study, 3, 5, 7, and 9 day old cultures were examined. The final concentration of cells attached to coverslips was approximately 200 cells per mm2. Cell Fixation Cells grown on coverslips for 3, 5, 7, or 9 days were removed from the culture dishes, rinsed 3 times in PBS (3 min each), and fixed in cold acetone for 10 min a t -20°C. After 5-10 min of air drying at 22”C, the coverslips were washed again in PBS 3 times for 3 min each and then stored at 4°C in excess PBS. lmmunofluorescent Microscopy For immunof luorescent staining of myofibrils, rabbit antiserum against porcine a-actinin (Transformation Research Inc.) was used. The specificity of the antibody was tested by immunoblotting. Samples of homgenates from adult heart and from tissue culture lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunostained with anti-a-actinin. These studies demonstrate that this antibody is specific for a-actinin a s we have reported previously (Isobe et al., 1988). Secondary antibodies were fluorescein conjugated goat anti-rabbit IgG (Organon-Teknika-Cappel). The protocol for staining the cells is as follows. The coverslips were removed from PBS and the attached 47 cells were incubated for 15 min. a t 37°C in 3% filtered nonfat milk to block nonspecific staining. This was followed immediately by a 60 min. incubation with antia-actinin diluted 1:20 with PBS. The cells were washed 3 times in PBS, blocked with 3% milk again, incubated with FITC-labelled goat anti-rabbit secondary antibody in a humid chamber a t 37°C for one hour or at 4°C for overnight. The preparations were washed in PBS and viewed with a Zeiss Universal light microscope equipped with epifluorescent illumination using a mercury light source. Photographs were taken on 35mm Kodak Plus-X film (ASA-125) a t 60 second exposure times. Electron Microscopy Electron microscopy was used to evaluate myofibrillar disarray and hypertrophy. Cultured cells were fixed in 2.5% glutaraldehyde, 2% formaldehyde, and 0.03% picric acid with 0.10 M cacodylate buffer pH 7.2 for 1or 2 h r at room temperature. After a brief buffer rinse, the cells were postfixed in 0.5% osmium tetroxide buffered to pH 7.2 with 0.10 M cacodylate buffer for 60 min a t 0°C. The cells were dehydrated in graded ethanols and embedded in Epon followed by polymerization at 60°C for 48 hr. The blocks were thin sectioned with a diamond knife, mounted on copper grids, and stained with lead citrate and uranyl acetate. Sections were viewed in a JEOL 1OOCX-I1electron microscope at a n acceleration voltage of 80 kV. QuantitativeAnalysis of Cytoplasmic Projections In order to obtain a n unbiased sampling of cells, 100 photographs of myocytes were taken at random from both normal and cardiomyopathic myocyte cultures at 3, 5, 7, and 9 days. The criterion for selecting the myocytes was determined by whether their myofibrils stained with anti-a-actinin antibody; fibroblasts were eliminated from the study by this criterion. Immunofluorescent microscopy was performed as previously described with a Zeiss Universal Microscope equipped with a Neofluor 63h.25 oil immersion objective and using a mercury light source. The 35 mm Kodak PlusX film negatives were printed using a direct method. Cell projections were defined as being longer than 3 mm on the 35 mm prints, that is, the projection on the cells that were measured and included in the study were longer than 5 pm in original size. All data were analyzed for significance by the Student’s t test and chi-square test; differences of P < 0.05 were considered to be significant. RESULTS Cardiac myocytes from both normal and cardiomyopathic hamsters are randomly distributed on the surface of the coverslips after 3 days. The myocytes appear round in shape (Fig. 1)and some of them are multinucleated. When cultured cardiomyocytes are stained with FITC-labeled anti-a-actinin, intense fluorescence is apparent in the Z bands of organized myofibrils in both normal and cardiomyopathic myocytes (CM). After 3 days in culture, the cell shape and Z band staining for individual myofibrils appears similar in normal and CM myocytes (Figs. 1,2). After 5 days in culture, the normal cardiac myocytes begin to form projections, which increase in both size and number with continuing development (Fig. 3). 48 J. LI AND L.F. LEMANSKI Fig. 1. Typical normal cardiomyocyte cultured 3 days and stained for a-actinin by a n indirect immunofluorescent method. The myocytes appear rounded in shape. The Z bands of the myofibrils are stained specifically. x 985. Fig. 3. Immunofluorescent staining pattern of a-actinin in a normal cardiomyocyte after 5 days in culture. The cardiomyocytes begin to form projections (arrows) and most of the myofibrils are arranged in parallel arrays. x 591. Fig. 2. Anti-a-actinin stained cardiomyopathic myocytes after 3 days in culture. The cell appears rounded in shape. Both Z band staining and cell shape appear similar to normal at this stage of development. ~ 9 8 5 . Fig. 4. Cardiomyopathic hamster heart myocyte cultured 5 days and stained for a-actinin. A disarray of myofibrils is obvious and most of the cells still have a somewhat rounded shape. The projections appear small (arrows). x 591. Well-formed myofibrils are oriented parallel to each other and to the longitudinal axes of the cells. By contrast, anti-a-actinin staining shows that myofibrils in some 5 day cultured cardiomyopathic cells are disoriented with respect to each other and fail to form the parallel arrays as in normal cells (Lemanski and Tu, 1983). Moreover, the cell projections are fewer and smaller in cardiomyopathic than in normal; in fact, some CM cells still appear to have a round shape (Fig. plex entanglements in the sarcoplasm. The shapes of most CM myocytes are still round after 7 days in culture (Fig. 6). Cells were cultured 9 days when myocytes and other cells appear to come in contact with each other, a phenomenon difficult to distinguish by phase contrast optics. Although some nonmuscle cells were similar in appearance to myocytes, FITC-labeled anti-a-actinin could be used to differentiate unequivocally between the cardiac myocytes and nonmyocytes in the population. Normal cardiomyocytes in culture show numerous projections in different directions (Fig. 7). CM cells have very few projections in culture, even though some of the CM cells have parallel myofibrils reminiscent of normal cells a t earlier times in culture (i.e., compare Figs. 5 and 8).These experiments were performed a total of 4 times, with virtually identical results in each case. 4). As the normal cell increases in size, there was a n increase in the number of projections, and after 7 days in culture, cells with a rounded shape are very rarely found (Fig. 5). The myofibrils in many of the CM cells are disarrayed abnormally after 7 days in culture. The disarray becomes even more pronounced during later development as many myofibrils appear to form com- a-ACTININ IN CARDIOMYOPATHIC HAMSTER HEART Fig. 5. Normal cardiomyocytes after 7 days in culture stained for anti-a-actinin. Projections increase in size and number as the cell size increases (arrows). x 675. 49 a-actinin. There are numerous projections in many different directions (arrows). x 591. Fig. 7. Normal cardiomyocyte cultured for 9 days and stained for Fig. 8. CM cardiomyocyte cultured for 9 days and stained for aactinin. The staining of individual myofibrils appears similar to that in normal control heart cells. Unlike normal cells, very few projections (arrow) form in CM cultures, even after 9 days. Those projections that do form in CM cells are generally smaller than in normal cells. x 591. Morphometric analyses of cell shape and cell projections were performed on developing cultured heart cells from both normal and cardiomyopathic hamsters. Data pertaining to this study together with the statistical analyses are presented in Fig. 9. First of all, we measured the projections that were defined as being longer than 5 Fm. To collect the data, 100 cells from normal and 100 cells from CM cultures at 3 , 5 , 7 , and 9 days were selected a t random from the culture plate. In normal cultures, the cytoplasmic projections showed a n obvious increase from 3 days to 5 days. Compared with normal, cardiomyopathic myocytes showed fewer projections during the same developmental period. By chisquare test analysis, there was a significant difference between normal and cardiomyopathic hamster heart cells at 3, 5, and 7 days in culture. No significant dif- ference was noted after 9 days in culture. Furthermore, when we analyzed the data obtained from the number of projections per cell, the difference was apparent. CM heart cells showed many fewer projections than normal. This result was shown to be statistically significant by the Student’s T test (Fig. 10). There was no significant change in the number of projections per cell during 3 or 5 days of culture; however, after 7 days in culture the difference between normal and CM cells was significant (2.3 to 1.5; P < 0.05). This difference became increasingly obvious after 9 days (3.8 vs 2.6; P < 0.01). Thus, there appeared to be a delay in the formation of projections in cultured CM cells when compared with normal. Myofibril disarray was obvious in CM hamster heart cells when examined in the electron microscope. These Fig. 6. CM cardiomyocyte stained for a-actinin after 7 days in culture. Myofibril disarray is obvious and some cells still have a rounded shape. A few short projections have formed in this cell (arrow). x 689. 50 J. LI AND L.F. LEMANSKI - @ cardiornyopathic I I I i 3 5 7 9 D a y s of incubation Fig. 9. Comparison of percentage of cells with projections (longer than 5 pm) from normal and cardiomyopathic hamster hearts in culture. By chi-square test analysis, there was a significant difference between normal and cardiomyopathic hamster heart cells at 3 , 5 , and 7 days (Pc0.05).There is no significant difference a t 9 days in culture. . ** 4 1 I @ I I I 1 3 5 7 9 Days of incubation Fig. 10. Comparison of numbers of projections per cell during the development in vitro from normal and cardiomyopathic hamster heart. Values are expressed as the mean t SEM; significant differences between control and CM animals are indicated by asterisks. *, P < 0.05; **, P < 0.01. observations corroborated the immunofluorescent data. Normal heart cells contained well-organized myofibrils that were in parallel arrangements with respect to each other (Fig. 111, while CM heart cells often contained disarrayed myofibrils (Fig. 12). DISCUSSION Our initial aim was to characterize the defects in heart of the cardiomyopathic hamster. In particular, we wished to discover what morphological differences, if any, were present between cardiomyopathic and normal hamster heart cells during development in vitro. We found that normal cardiomyocytes begin to have cytoplasmic projections after 5 days in culture, and increasing numbers with increasing time in culture. These projections probably represent a continuation of heart differentiation. It is clear that hamster heart cells have not completed differentiation at birth. For example, the T-sysiem does not appear in vivo until 4 or 5 days post partum (Colgan et al., 19781, interestingly, the same time span i t takes for cultured cells to form projections. By 9 days in culture, most normal cells showed several projections that spread out in different directions. We consider it possible, that if these projections were in vivo, they would extend out to form intercellular connections (i.e., intercalated discs) with other cells. By contrast, cardiomyopathic myocytes form fewer projections, which appear later than normal in culture, in fact, many CM cells had not formed projections, even as late a s 9 days in culture. Unlike skeletal muscle, cardiac muscle is not a syncytium, but is composed of individual branching cells containing many parallel myofibrils. The cells of cardiac muscle are joined together by intercalated discs composed of specialized junctions that permit both mechanical and ionic coupling between cells so as to allow integrated function. Our results showing a substantial reduction in the size and number of projections in cultured CM cardiomyocytes. However, Sorenson et al. (1985)found that enzymatically isolated ventricular myocytes from adult CM hamster were wider and longer than normal. Recent immunofluorescent and electron microscope studies in our laboratory (Luque e t al., 1989, 1990) using antibody against the gap junction peptide, Connexin-43 (a gift from Dr. Eric C. Beyer, Beyer et al., 1989)showed CM cells to have fewer gap junctions and a more diffuse staining pattern for gap junctions than normal. While this is a most interesting correlation, further work will be required to determine the precise nature of these abnormalities. Since adhesion to the substrate seems to be required for “normal” cell growth in tissue culture, the lack of normal projections in CM hamster myocardial cells may relate to abnormal focal cell adhesion sites. In a preliminary study in our laboratory, Osinska et al. (1988) found that CM cardiomyocytes attached to plastic culture dishes at a lower than normal efficiency; our data showed that a significantly lower percentage of CM cells, as compared with normal, attached a t the various time intervals examined. Whether the abnormalities in attachment and myofibril organization in CM cells are in any way related to abnormal focal adhesions and/or extracellular matrix receptors requires further study. It is well known that oncogenic cell transformation results in altered adhesive properties of the transformed cell (Burridge, 1986). This, in turn, affects their internal cytoskeletal organization a s well a s their morphology. In very recent studies, Borg et al. (1990) demonstrated that focal adhesions containing vinculin are formed in cultured cardiomyocytes in association with the assembly of myofibrils; the adhesions co-localize with extracellular matrix (ECM) receptors on cultured cardiomyocytes. In the present study, anti-a-actinin was used to stain the myofibrils. The idea that a-actinin possibly may have a membrane attachment function has been revived because of two recent reports. First, a-actinin was shown to have a n affinity for particular membrane-associated lipids and these lipids were found to enhance the interaction of a-actinin with actin (Burn e t al., 1985). Secondly, ol-actinin was reported to bind to vinculin (Craig, 1985). Other than a somewhat abnormal arrangement of myofibrils and lack of projections Fig. 1I . Electron micrograph of a normal cardiomyocyte after 7 days in culture. The myofibrils (MF) show a parallel alignment relative to each other with many of the 2-bands (Z) in register. x 21,420 Fig. 12. Electron micrograph of a cardiomyopathic cardiomyocyte after 7 days in culture. The myofibrils (MF) are disoriented with respect to each other. 2, 2 line. x 30,800 in CM cells, a-actinin localization of individual myofibrils did not appear to differ from normal. One possible explanation for the myofibril disarray observed in CM heart cells (Lemanski and Tu,1983)is that sarcomeric organization is delayed in myocytes of the cardiomyopathic hamster. Relevant to this and 52 J. LI AND L.F. based upon our earlier studies, the disoriented myofibrils show irregular arrays and overlap each other (Figs. 4,6). This phenomenon may be related to the CM cells’ inability to spread out and form projections as normal cells do. In addition to a potential abnormality in adhesion sites, another possible explanation of the abnormal cell shapes for CM heart cells could relate to their altered ability to bind calcium by the sarcoplasmic reticulum (Pearce et al., 1985; Jasmin and Proschek, 1984). Moreover, since swollen and pleomorphic mitochondria are regarded as one of the features of the aberrant cardiac cells in hamsters (Hoppel et al., 1982; Wikman-Coffelt et al., 1986), i t is possible that this may affect the formation of the projections in the cardiomyopathic myocytes. Since mitochondria transform the energy of metabolites into available energy that is useable by the cell, the aberrant mitochondria may affect the energy involved in forming the projections andlor aligning the myofibrils. In addition, projections in “normal” cells, which we believe mimic in vivo branching, undoubtedly have a n important role in the formation of intercalated discs between cells. It is possible that a deficiency of normal branching in cardiomyocytes could influence the formation of junctions between cells, which in t u r n would affect ion transfer a s well as metabolism. It has been found that cardiomyopathic hamsters accumulate excess calcium in their hearts. In fact, the concentration of calcium in the left ventricles of CM animals 60 days of age is, astonishingly, 14-fold higher than that in normal (Ma and Bailey, 1979). If CM heart cells develop abnormal membranedmembrane junctions, then it is possible that high levels of Ca2+ might accumulate in the CM cells. At the moment, the central question of which protein or proteins might be involved in the abnormal projections in CM cells remains unanswered, because there are several proteins associated with attachment sites a t the cell periphery. Studies to date concerning adhesion sites reveal that such structures are associated with several cytoskeletal proteins including actin, talin, fimbrin, and vinculin (Burridge and Connell, 1983a,b; Hilenski et al., 1989; Terracio et al., 1989). Major research objectives in future work on cardiomyopathic hamster heart cells will be to identify the elements involved in the focal adhesion sites and transmembrane linkages between the extracellular matrix and intracellular cytoskeletal elements and to attempt to understand the cell and molecular mechanism( s) responsible for myofibril disarray and the abnormal cell projections. ACKNOWLEDGMENTS This study was supported by NIH grants HL 32184 and HL 37702 and a grant from the American Heart Association to L.F.L. The authors wish to thank Dr. Douglas Robertson for his assistance with statistical analysis and Dr. Yuji Isobe and Dr. Hanna Osinska for their excellent advice on techniques. We are grateful to Sharon Lemanski for technical help in providing normal and CM hamsters a t the proper ages for the matched cultures, Masako Nakatsugawa for assistance with the darkroom work, and Jo-Ann Pellett and Nancy Snyder for typing the manuscript. LITERATURE CITED Beyer, E.C., J. 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