Time course of the apoptotic cascade and effects of caspase inhibitors in adult rat ventricular cardiomyocytes.

K. Suzuki, S. Kostin, V. Person, A. Elsässer and J. Schaper. Time Course of the Apoptotic Cascade and Effects of Caspase Inhibitors in Adult Rat Ventricular Cardiomyocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 983-994. Interpretation of the rate of apoptosis in diseased hearts is hampered by the fact that the time course of the apoptotic cascade in adult cardiomyocytes is largely unknown. Therefore, we established a standardized in vitro system, relevant to the in vivo situation of heart failure, using adult de- and redifferentiating cardiomyocytes to determine the time intervals necessary for the different steps of the apoptotic cascade to occur. Apoptosis was induced with 0.1 mmol/l H(2)O(2)in adult rat cardiomyocytes 10 days in culture. Dosages >0.5 mmol/l H(2)O(2)produced necrosis. Disruption of the mitochondrial membrane potential (Deltapsim) was the earliest sign of apoptosis and occurred at 2 h after H(2)O(2)exposure. The number of annexin V (translocation of phosphatidylserine) and PhiPhiLux (activation of caspase-3) positive cells significantly increased after 4 h and remained constant thereafter. Bcl-2 levels decreased. At 9 h, Bax expression was significantly elevated resulting in a reduced Bcl-2/Bax ratio. DNA fragmentation detected by TUNEL and ssDNA peaked at 14 h, parallel to the appearance of apoptotic ultrastructural changes. Although DNA fragmentation was inhibited by zVAD-fmk, Ac-DEVD-CHO, zLEVD-fmk, these caspase inhibitors failed to inhibit disruption of Deltapsim and increased the number of necrotic cells. Catalase inhibited both apoptosis and necrosis. Our results indicate that the occurrence of the different steps of the apoptotic cascade is time-dependent and tightly regulated. Caspase inhibitors reduce apoptosis but increase the rate of necrosis, suggesting that the cells are destined to die upstream of the caspase step, i.e. by mitochondrial damage. These data provide the basis for the critical evaluation and interpretation of the occurrence of apoptosis in failing hearts. Copyright 2001 Academic Press.