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From the Second Department of Internal Medicine,*
Gifu
University School of Medicine, Gifu; and the Department of Food
Science,
Kyoto Women’s University,
Kyoto, Japan
Dynamic process of apoptosis has not been elucidated in adult rat cardiomyocytes. Soluble Fas ligand (0.1 µg/ml) in the presence of actinomycin D (0.05 µg/ml) induced apoptosis in cultured adult rat cardiomyocytes, as documented by activated caspase-3, DNA fragmentation, and apoptotic ultrastructure. In the present model, we observed 60 adult cardiomyocytes with a normal rod shape under a real-time videomicroscope continuously for 48 hours. Seventeen cells (28%) were unchanged and 7 cells (12%) showed oncosis (so-called necrosis) in which no beating was evident. In the remaining 36 cells (apoptosis, 60%), a slow beating (17 ± 3/min) was initiated 16 ± 1 hours later. Approximately 1 hour later, the rod cells showed long-axial shortening as bone- or club-like, or square-shaped, accompanied with faster beating rates (35 ± 7/min). In 29 cells (type A1 and A2), marked shrinkage occurred; the cellular shape became almost completely round with a smooth surface and the beating ceased 3.0 ± 0.4 hours later. Then, smooth budding appeared 0.6 ± 0.2 hours later. Apoptotic bodies were found in 8 cells 10 ± 4 hours later (type A1, 13%) but not in 21 cells (type A2, 35%). In the other 7 cells (type A3, 12%), the cell surface became rough 8 ± 3 hours later and the beating ceased. Maximal beating rate was greatest in type A1 (72 ± 26/min) and greater in type A2 (29 ± 5/min) than in type A3 (10 ± 2/min). Electron microscopy confirmed apoptotic ultrastructure even in the cardiomyocytes with bone-, club-like, or square shapes, suggesting that type A3 as well as A1 and A2 is also under apoptotic process. A caspase inhibitor, zVAD.fmk, blocked beating, apoptotic morphology, and DNA fragmentation, indicating these depended on caspase activation. In the caspase-dependent apoptotic process of cultured adult cardiomyocytes, beating and the following deformity of the cellular edges were the initial signs and the rate of beating was related with the subsequent three different processes of apoptosis.
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