Figure 1.

Contributions of waste metabolic buildup to apoptosis induced by chronic hypoxia. (a and b) Parallel cultures of cardiac myocytes were exposed to hypoxia as described in Methods. In a, the medium was replaced with fresh hypoxic medium every 12 hours; in b, there was no medium replacement. Cultures were harvested at the indicated times and processed for DNA fragmentation. (c) Intracellular ATP, medium glucose, and [pH]_o were measured in parallel cultures as described in Methods; results are means of 3 separate experiments. Open circles are results from cultures without medium replacement; closed circles, with medium replacement. (d–f) Typical fields of myocytes stained with HOECHST 33342 and anti-myosin antibody as described in Methods. (g) Quantitations of HOECHST-stained condensed nuclei, also described in Methods. At 24 and 48 hours, less than 2% of cells were PI positive (scored as necrotic) under any condition; at 72 hours, hypoxia-acidic cultures had more PI-positive cells, and these were scored as apoptotic if they contained condensed nuclei. Costaining populations were not distinguished from PI-excluding cells at this stage. Results are representative of at least 3 experiments.