Immunocytochemical analysis of the regeneration of myofibrils in long-term cultures of adult cardiomyocytes of the rat
| Autor: | Marcus C. Schaub, Monika E. Eppenberger, Hans M. Eppenberger, Ingrid Hauser, Thomas Baechi, Claude A. Dechesne, Ulrich T. Brunner |
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| Rok vydání: | 1988 |
| Předmět: |
Immunocytochemistry
Fluorescent Antibody Technique Muscle Proteins Video microscopy Myosins Biology Sarcomere medicine Animals Regeneration Connectin Creatine Kinase Molecular Biology Cells Cultured Myomesin Myocardium Antibodies Monoclonal Colocalization Heart Cell Biology Anatomy Rats Cell biology Cell culture Collagenase biology.protein Carrier Proteins Myofibril Developmental Biology medicine.drug |
| Zdroj: | Developmental Biology. 130:1-15 |
| ISSN: | 0012-1606 |
| DOI: | 10.1016/0012-1606(88)90408-3 |
| Popis: | Dissociated adult rat ventricular cardiomyocytes obtained from hearts by retrograde perfusion with collagenase were investigated in long-term cultures. Myofibril regeneration, isoprotein transition of alpha- and beta-myosin heavy chain (MHC), and M-band localization of M-creatine kinase in the reconstituting heart cells were studied. Myofibril formation was demonstrated by the use of antibodies against either cardiac C-protein or myomesin as early differentiation markers. Four days after plating, small myofibrils could be identified in attached cells in a perinuclear fashion; later in culture the cells displayed various shapes and myofibril distribution. Frequently a patchy distribution of myofibrils within the extending peripheral processes could be observed. Colocalization of sarcomeres and phalloidin-stained F-actin filament bundles was demonstrated by double fluorescence staining and by the use of high intensifying video microscopy and computerized image processing. The immunofluorescence distribution of alpha- and beta-MHC isoproteins in newly isolated and cultured cardiomyocytes changed from 100% alpha-MHC and 70% beta-MHC in rod-shaped cells to about 100% beta-MHC and 70% alpha-MHC in spread out cultured cells. This shift was corroborated by a relative gradual decline in alpha-MHC at the expense of increasing amounts of beta-MHC with time in culture as assessed by sodium dodecyl sulfate gel electrophoresis of total cell homogenates. In addition, whereas rod-shaped newly isolated cardiomyocytes showed a clear M-band association of M-creatine kinase as found in adult heart tissue, adult cultivated spread out cells did not show a cross-striated pattern after incubation with antibody. Taken together, these observations suggest that adult cardiomyocytes not only undergo extensive morphological transitions in long-term cultures, but also generate new myofibrillar structures lacking M-creatine kinase and containing the beta-MHC, thus fitting the characteristics of fetal myofibrils. These results indicate a change from the adult terminally differentiated to a less differentiated state of the cardiac cells in culture. |
| Databáze: | OpenAIRE |
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