Figure 2.
Activation of the caspase transgene in vivo results in massive cardiac myocyte apoptosis and death of the animals. (a) Mortality following intraperitoneal administration of vehicle alone (81.9% polyethylene glycol 400, 9.1% Tween-80, 9% dimethylacetate) or FK1012H2 (30 mg/kg in the vehicle) to 3-week-old WT mice and transgenic line 7, 169, and C360A mice. Animals were observed for death for 7 days following FK1012H2 administration. (b) Inverse dose-dependence of the time to death following intraperitoneal administration of the indicated doses of FK1012H2 to WT or transgenic line 7 (TG) mice. Animals were observed for death for 7 days following FK1012H2 administration, with no deaths occurring in the WT group. (c) Caspase inhibition delays time to death following transgene activation. Vehicle (0.9% saline) or the polycaspase inhibitor IDN 1965 (12 mg/kg in vehicle) was administered intraperitoneally to line 7 transgenic mice 45 minutes before FK1012H2 (30 mg/kg intraperitoneally) and every 4 hours thereafter as indicated by the triangles. Bars represent the survival times of individual animals that received vehicle or IDN 1965. Animals were observed for death until all had died. *P < 0.0001. (d) Activation of the transgene caspase by FK1012H2. Immunoblot of cardiac homogenates from WT and transgenic line 7, 169, and C360A mice treated 1.5 hours earlier with vehicle or FK1012H2 (30 mg/kg intraperitoneally). Processing of procaspase-8 is indicated by disappearance of the uncleaved moiety; cleavage fragments are not reliably detected in tissue homogenates, presumably because of rapid degradation. The lower portion of the blot was reacted with an antibody against mouse tubulin as a loading control. (e) Induction of cardiac apoptosis by FK1012H2. Genomic DNA from the hearts of WT and transgenic line 7 mice 1 hour after the administration of vehicle or FK1012H2 (30 mg/kg i.v.) was size-fractionated on an agarose gel containing ethidium bromide. (f) TUNEL analysis of FK1012H2-induced apoptosis. Paraffin-embedded sections of hearts from WT and line 7 transgenic mice 1 hour after administration of vehicle or FK1012H2 (30 mg/kg i.v.). TUNEL-positive cells were primarily myocytes, but additional unidentified cells were also present that may represent degenerating myocytes or infiltrating inflammatory cells due to magnitude and rapidity of the death. Bar, 20 μM.