Characterization of ultrastructure and its relation with DNA fragmentationin Fas-induced apoptosis of cultured cardiac myocytes

The purposes of the present stud, were to define precisely the ultrastructu ral features of apoptosis in cultured cardiomyocytes and to determine wheth er DNA fragmentation is essential for the apoptotic morphology. When cultur ed neonatal murine cardiomyocytes were incubated with an agonistic anti-Las antibody in the presence cycloheximide, approximately 70%, of them had los t their viability after 24 h, The dead cardiomyocytes showed the typical ul trastructural changes of apoptosis on transmission and scanning electron mi croscopy. as well as by positive in situ nick end-labelling (TUNEL), positi ve Taq polymerase-based in situ ligation, a DNA ladder pattern on gel elect rophoresis, and an increase in the active fragment of caspase-3, According to TUNEL at the electron microscopic level, apoptotic nuclear change, cytop lasmic shrinkage, and DNA fragmentation always occurred simultaneously in a poptotic cardiomyocytes, Other ultrastructural features of apoptosis were t he appearance of abundant lipid-like structures in the cytoplasm of cardiom yocytes at the early phase, and a high incidence of plasma membrane rupture and formation of apoptotic bodies at the later phase. When zinc, an inhibi tor of Ca2+/Mg2+-dependent endonuclease, was added to the present model, ac tivation of caspase-3 and an apoptotic ultrastructure were still observed i n spite of the lack of DNA fragmentation, indicating that this type of myoc yte death is also apoptosis, In conclusion, the typical apoptotic ultrastru cture and DNA fragmentation occur simultaneously in association with caspas e-3 activation in Fas-stimulated cultured cardiomyocytes. Apoptotic morphol ogy can, however, be observed even without DNA fragmentation, Copyright (C) 2000 John Wiley & Sons, Ltd.