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First published online August 6, 2004
Journal of Experimental Biology 207, 3189-3200 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01109

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A unique pathway of cardiac myocyte death caused by hypoxia–acidosis

Regina M. Graham, Donna P. Frazier, John W. Thompson, Shannon Haliko, Huifang Li, Bernard J. Wasserlauf, Maria-Grazia Spiga, Nanette H. Bishopric and Keith A. Webster^*

Department of Molecular and Cellular Pharmacology and the Vascular Biology Institute, University of Miami Medical Center, Miami, FL 33101, USA

View larger version (16K): [in a new window]   Fig. 1. Nuclear condensation and caspase activity during chronic hypoxia. (A) Rapidly contracting neonatal cardiac myocytes were exposed to hypoxia (PO2 <8 mmHg) in minimal essential medium (MEM) with 5% fetal calf serum (FCS) as described previously (Webster and Bishopric, 1992). The medium was replaced every 12 h with fresh hypoxic medium so that the pH was maintained at >7.2, glucose was not depleted and ATP level was maintained at >70% control. Nuclei were stained with Hoechst 33342 and examined microscopically as described previously (Dougherty et al., 2002; Webster et al., 1999). (B) Cultures were maintained as in A, harvested as indicated, and the cells lysed for caspase assays using a kit from Ambion Inc. (Austin, TX, USA) as described previously (Dougherty et al., 2002). Differences were not significant at P=0.30 (N=6). Values are means ± S.E.M.

View larger version (49K): [in a new window]   Fig. 2. Activation of programmed death by waste metabolites under chronic hypoxia. (A,B) Parallel cultures of cardiac myocytes were exposed to hypoxia. In A, the media was replaced with fresh hypoxic medium every 6 h. In B, the media was not replaced. 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 (Webster et al., 1999); results are means of three separate experiments; squares represent results from cultures with media replacement; open circles show results without media replacement; filled circles represent the 72 h aerobic control. (D–F) Typical fields of myocytes stained with Hoechst 33342 and anti--MHC antibody (Webster et al., 1999). 1G Quantitations of Hoechst-stained condensed nuclei were as described in Webster et al. (1999. Results are representative of at least three experiments.

View larger version (24K): [in a new window]   Fig. 3. Induction of apoptosis by conditioned medium. Cultures were grown under hypoxia with or without medium change. After 48 h, the medium was removed, and the cells were analyzed for DNA fragmentation (top panel). The conditioned media was centrifuged at 800 g for 5 min to pellet cells and debris, added directly to a second set of plates, and these were incubated in air or under hypoxia as indicated. After 24 h, these cells were also harvested and analyzed for DNA fragmentation. [pH]o was measured in all cases immediately before harvesting the cells. Note that the control samples shown in the last two lanes of the bottom left panel did not receive conditioned medium. Results are representative of three separate experiments.

View larger version (27K): [in a new window]   Fig. 4. [pH]o neutralization prevents apoptosis. Conditioned medium was generated as described in Fig. 2. (A) The top panel shows DNA ladders from the cardiac myocytes used to generate the conditioned medium. In the bottom panel, the conditioned medium was added directly to fresh plates of cardiac myocytes (middle two lanes) or it was neutralized to pH 7.4 with Hepes (20 mmol l–1 final concentration) and NaOH and then added to a second set of fresh cardiac myocytes. Both sets of plates were incubated under hypoxia for 24 h and analyzed for DNA fragmentation. Control plates were incubated under aerobic or hypoxic conditions in parallel. (B) Parallel sets of cardiac myocytes were exposed to hypoxia without medium change; in the first set (left panel), the acid was allowed to accumulate exactly as described in Fig. 2B; in the second set (right panel), experimentally predetermined aliquots of Hepes and NaOH were added every 12 h to maintain a [pH]o of 7.2.

View larger version (48K): [in a new window]   Fig. 5. (A) Analysis of mRNA and protein from aerobic and hypoxic (24 h) cardiac myocytes. Parallel plates of cardiac myocytes were cultured under aerobic or hypoxic conditions for 24 h and then harvested for RNA extraction. RNA was purified and reverse transcribed by standard procedures. Differentially labeled cDNAs were hybridized to arrays of 20 000 specific gene sequences (known and unknown cDNAs and expressed sequence tags) from combined rat and mouse libraries (Incyte Inc., Freemont, CA, USA). Data were analyzed using Excel 98 software. Hypoxia-regulated marker genes including heme oxygenase (HO), glucose transporter (GLUT), pyruvate dehydrogenase kinase (PDK), triosephosphate isomerase (TPI), tyrosine amino transferase (TAT) and metallothionein (MT-1) are shown. (B) Northern blot of cardiac myocyte RNA extracted from hypoxic cultures. The top gel shows BNIP3; the bottom gel shows ß-actin. (C) Western blot analysis of proteins from hypoxic cardiac myocytes as in A. Anti-BNIP3 recognizes two bands at 60 kDa and 30 kDa, corresponding to SDS-resistant homodimers and monomers, respectively. Lower panels show the same blot probed with anti-Bax, Bak and ß-actin. (D) Rat hearts were removed and perfused by the Langendorf method, as described previously (Webster et al., 1999). Hearts were subjected to no flow for 1 h or to perfusate equilibrated with 100% N2 for 2 h. RNA was analyzed by northern blot, as above.

View larger version (55K): [in a new window]   Fig. 6. Association of BNIP3 with subcellular fractions. Cardiac myocytes were subjected to hypoxia as described in Fig. 1. At the indicated times, cells were harvested, rinsed and lysed (Kubasiak et al., 2002). Lysed cells were untreated (left panels) or subjected to alkaline solubilization (right) (Kubasiak et al., 2002). The pH of each sample was verified by sampling small aliquots. After treatments, samples were separated into subcellular fractions and analyzed by western blots. Blots were stripped and re-probed with anti-succinate dehydrogenase (Upstate Biotechnology, Lake Placid, NY, USA) probes to define the purity of fractions. Results are representative of two separate experiments.

View larger version (29K): [in a new window]   Fig. 7. Characteristics of programmed death by BNIP3. (A) Cardiac myocytes were subjected to hypoxia–acidosis as described in Fig. 2. At the indicated times, samples of media were taken for analysis of lactate dehydrogenase (LDH) activity (open circles), or plates were stained with Trypan blue (filled circles) (Kubasiak et al., 2002). Data are expressed as % of cells stained with Trypan blue or % LDH released relative to total LDH in homogenates. (B) Cardiac myocytes were subjected to hypoxia–acidosis in the absence or presence of the broad-range caspase inhibitor Boc-D as indicated. Staurosporine (Sta; 0.1 µmol l–1 for 8 h) is shown as a positive control.

View larger version (64K): [in a new window]   Fig. 8. (A) Cardiac myocytes were subjected to hypoxia–acidosis in the absence or presence of the mitochondrial permeability pore (MPTP) inhibitors bongrekic acid (BA) or decylubiquinone (DUB), as indicated. (B) Cardiac myocytes were exposed to normoxic or hypoxia–acidosis conditions. At the times indicated, cells were loaded with MitoTracker Red dye and analyzed by confocal microscopy as described (Kubasiak et al., 2002). Arrows indicate intense staining around nuclei in aerobic myocytes and reduced staining under hypoxia. Results are representative of three experiments.

View larger version (18K): [in a new window]   Fig. 9. Pathway of cardiac myocyte death by exposure to hypoxia–acidosis. Hypoxia mediates accumulation of hypoxia-inducible factor-1 (HIF-1), and activated HIF-1 induces transcription of BNIP3. BNIP3 is loosely membrane bound at neutral pH but translocates into membranes, including mitochondria, when the pH decreases. Acidosis is caused by anaerobic metabolism. BNIP3 stimulates opening of the mitochondrial permeability transition pore (MPTP), releasing pro-apoptotic factors including apoptosis inducing factor (AIF), cytochrome c and calcium. Myocyte death, involving DNA fragmentation and nuclear condensation, requires MPTP opening but does not involve activation of caspases. The black box indicates that the proteases and DNases, presumed to be involved in cell death, are not yet identified. Broken lines indicate points where the death pathway can be blocked. Bongrekic acid (BA) and decylubiquinone (DUB) both inhibit MPTP opening and block myocyte death.