Immunocytochemical analysis of the regeneration of myofibrils in long-term cultures of adult cardiomyocytes of the rat

Abstract

Dissociated adult rat ventricular cardiomyocytes obtained from hearts by retrograde perfusion with collagenase were investigated in long-term cultures. Myofibril regeneration, isoprotein transition of α-and β-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 α- and β-MHC isoproteins in newly isolated and cultured cardiomyocytes changed from 100% α-MHC and 70% β-MHC in rod-shaped cells to about 100% β-MHC and 70% α-MHC in spread out cultured cells. This shift was corroborated by a relative gradual decline in α-MHC at the expense of increasing amounts of β-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 β-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.