Characteristics of death of neonatal rat cardiomyocytes following hypoxia or hypoxia-reoxygenation: the association of apoptosis and cell membrane disintegrity.

Irreversibly injured cardiomyocytes are positive for terminal deoxynucleotidyl transferase nick end-labeling (TUNEL), making it controversial as to whether TUNEL-positive cardiomyocytes induced by hypoxia-reoxygenation are apoptotic (secondarily necrotic) or primarily necrotic. We investigated the relationship between plasma membrane integrity and DNA fragmentation in hypoxic-reoxygenated cardiomyocytes. Cardiomyocytes were prepared from neonatal rat heart and exposed to hypoxia. The plasma membrane integrity was assessed by propidium iodide (PI) staining. The mode of DNA fragmentation was assessed by TUNEL and in situ polymerase chain reaction ligation assay. Furthermore, caspase-3 activity was measured in hypoxic-reoxygenated cardiomyocytes. Reoxygenation for 24 h after 3-8 h of hypoxia increased TUNEL positivity. However, the appearance of PI-positivity preceded that of TUNEL at various time points following reoxygenation. In contrast, TUNEL-positive but PI-negative cells were rarely found. In the hypoxic-reoxygenated cells, caspase-3 activity was increased, and PI- and TUNEL-positive cardiomyocytes possessed a sufficient number of double-strand DNA breaks with single-base 3'-OH terminals. In cardiomyocytes subjected to hypoxia-reoxygenation, the appearance of TUNEL positivity was delayed in comparison to membrane disintegrity, but in these cells caspase-3 has been activated and the mode of DNA fragmentation was apoptosis-specific. Thus, hypoxia-reoxygenation induces apoptosis associated with cell membrane disintegrity in cardiomyocytes.