Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53 (original) (raw)
Correlation of hypoxia-mediated apoptosis with change in extracellular pH. We reported previously that rapidly contracting cardiac myocytes remained fully viable and contractile during culture under severe hypoxia for up to 6 days (48). Glycolysis was induced approximately 10-fold within 1 hour, and there was no evidence of major cell loss. The intracellular ATP of hypoxic myocytes dropped to about 70% of control plates, and a slightly reduced contractility correlated with lower intracellular cAMP in the hypoxic cultures. These results contrast with more recent reports of significant cardiac myocyte cell loss by apoptosis after 48–72 hours of exposure to an equivalent degree of hypoxia (44, 45). To investigate this apparent discrepancy, we exposed cultures of myocytes to 2 different hypoxic regimens and measured the levels of apoptosis by DNA ladders and HOECHST staining. In the first set, the cultures were maintained continuously under hypoxia, and the medium was replaced twice daily with fresh hypoxic medium to prevent the buildup of waste metabolites (48). In the second set, the cultures were maintained in parallel, but the medium was not replaced. The cultures were also monitored for ATP, glucose, and extracellular pH ([pH]o) as described previously (48, 58) and in Methods. The results are shown in Figure 1, a and b. There was no evidence of DNA fragmentation under the first set of conditions (Figure 1a), but cultures subjected to hypoxia without medium changes showed significant DNA laddering after 48 hours and extensive laddering after 72 hours (Figure 1b). In agreement with our previous observations, when the medium was replaced twice daily, ATP levels dropped to about 75% of aerobic control levels after 24 hours and remained stable thereafter; [pH]o did not change, and glucose levels remained high (Figure 1c). Under these conditions, the myocytes continued to contract for the duration of the experiment, as reported previously, and there was no significant loss of total DNA or protein (data not shown; ref. 48). When the medium was not replaced, intracellular ATP levels were sustained up to 48 hours but dropped dramatically at 72 hours, coincident with the loss of most of the cells by apoptosis. Glucose declined progressively over the 72-hour period but was not depleted, and [pH]o declined steadily to a final value of about 6.0 (Figure 1c). Under these conditions, contractions ceased before 48 hours (data not shown).
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.
Cardiac myocytes were grown on glass coverslips and were fixed and double stained with HOECHST 33342 and anti-myosin antibody (Figure 1, d–f). In cultures grown aerobically, abundant myofilaments with clear cross-striations were evident with the myosin stain (Figure 1d, right panel). As indicated by the white arrows, most of the nuclei were oval, and there was sparse evidence of condensation or internal fragmentation. In this field, 29 nuclei were scored normal and 1 was condensed; 25 nuclei were localized within cells that were myosin positive. At 72 hours, the unfed hypoxic cultures (Figure 1e) still stained strongly with myosin antibody, but there was clear deterioration of the myofilaments, and cross-striations were no longer clearly visible. In the field shown, 22 nuclei were scored condensed (examples are indicated by the arrows), and 14 were normal; only 2 nuclei in this field were localized to myosin-negative cells. In contrast, cells that received medium replacement still exhibited myofilaments with intact cross-striations after 72 hours of hypoxia (Figure 1f, right panel, arrow at far right), and most of the nuclei were normal. In this field, 27 nuclei were scored normal and 3 condensed; 2 nuclei were localized to nonmyocytes. White arrows indicate normal cardiac myocyte nuclei; the pink arrow indicates a condensed nucleus.
These data were quantitated as shown in Figure 1g. The bar graphs indicate the percentage of apoptotic nuclei scored for each condition. Control aerobic cultures contained 5–7% apoptotic cells, similar to previous reports (51). This increased to 44% after 48 hours of hypoxia with metabolite buildup, and to 60% after 72 hours of hypoxia. In hypoxic cultures without metabolite buildup, the apoptotic index was significantly lower (11 ± 1.5% at 72 hours). This slight increase in apoptosis over aerobic myocytes could be due to transitory acidosis under these conditions or to other factors associated with hypoxic incubation. Except for the 72-hour hypoxia-acidosis condition, necrotic cells (PI-positive noncondensed nuclei) were less than 5% of the total cells counted (see Methods; data not shown).
These results suggest either that proapoptotic factors accumulate in the medium during hypoxia or that vital components are depleted. To test these possibilities, medium from 48-hour hypoxic cultures that were just beginning to show signs of DNA laddering (Figure 2, top) was transferred to fresh cardiac myocytes. These cells were incubated for 24 hours under either hypoxic or aerobic conditions. Apoptosis was monitored by DNA fragmentation. Control plates received medium from hypoxic cells that underwent medium replacement (+ med). A typical experiment is shown in Figure 2. In this case, significant apoptosis was apparent in both aerobic and hypoxic cultures 24 hours after exposure to the spent medium. Glucose and ATP levels were maintained in all cultures (data not shown). The [pH]o in the aerobic plates remained stable at 7.0, whereas the [pH]o under hypoxia dropped to 6.1. More DNA fragmentation appeared in the sample from the 24-hour hypoxic plate, correlating with the lower [pH]o. In control plates (Figure 2, bottom right), there was only slight DNA laddering at 24 hours, and the [pH]o remained high in these cultures.
Induction of apoptosis by conditioned medium. Cultures were grown under hypoxia with or without medium change. After 48 hours, the medium was removed and the cells were analyzed for DNA fragmentation (top). The spent medium was centrifuged at 800 g for 5 minutes, to pellet cells and debris, and was added directly to a second set of plates. These plates were incubated in air or under hypoxia as indicated. After 24 hours, these cells were also harvested and analyzed for DNA fragmentation. Controls show untreated cells harvested at the time of medium change. [pH]o was measured in all cases immediately before harvesting the cells. Control samples shown in the last 2 lanes of the bottom left panel did not receive spent medium. Results are representative of 3 separate experiments.
The induction of apoptosis of fresh cardiac myocytes by spent medium could be due to the accumulation or depletion of factors in this medium. Obvious candidates for proapoptotic factors are the protons extruded from the hypoxic cells. To determine whether this was the case, the pH of the spent medium from 48-hour hypoxic cultures was readjusted to 7.6 with NaOH and HEPES before adding back to fresh cardiac myocytes. Apoptosis was again monitored in the recipient cells as described in Figure 2. These results are shown in Figure 3a. As before, acidic spent medium from hypoxic myocytes again caused extensive apoptosis after 24 hours (Figure 3a, bottom, lanes 4 and 5). In contrast, minimal DNA fragmentation was detected in the control cells (normal medium, lanes 2 and 3) and in samples from cells exposed to the same spent medium after pH neutralization (lanes 6 and 7). This suggests that an acidic pH is important for the induction of nuclear fragmentation by spent medium from hypoxic cultures. It also suggests that acidic [pH]o may directly induce apoptosis. To test this hypothesis directly, parallel cardiac myocyte cultures were again exposed to hypoxia as described in Figure 1. In the first set, the medium was not replaced and became acidic (conditions were the same as described in Figure 1b). In the second set of cultures, the medium was not replaced, but [pH]o was maintained higher than 7.0 by adding predetermined amounts of HEPES and NaOH at 12-hour intervals. The results are shown in Figure 3b. In the absence of additional buffer, [pH]o dropped to 5.7 at 48 hours, and there was extensive apoptosis at both 48 hours and 72 hours. In the cultures with neutralized medium, DNA fragmentation was significantly reduced.
Neutralized medium prevents apoptosis. Conditioned medium was generated as described in Figure 2. (a, top) DNA ladders from the cardiac myocytes used to generate the spent medium. (a, bottom) Spent medium was added directly to fresh plates of cardiac myocytes (middle 2 lanes), or it was neutralized to pH 7.4 with HEPES (20 mM 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 hours and analyzed for DNA fragmentation. Control plates were incubated under aerobic or hypoxic conditions in parallel. (b) Parallel sets of cardiac myocytes exposed to hypoxia without medium change. In the first set (b, left), the acid was allowed to accumulate exactly as described in Figure 1b; in the second set (b, right) aliquots of 250 mM HEPES and 250 mM NaOH were added every 12 hours to maintain a [pH]o of approximately 7.1. Measurements of DNA fragmentation and determinations of percent condensed nuclei (c) were as described in Methods. In all cases, the medium pH was measured immediately before the cultures were harvested. (d) Typical field of myocytes stained with HOECHST 33342 and anti-myosin antibody as described in Figure 1d. Results in a, b, and c are from typical experiments; error bars in c are SEM (n = 3).
These results were confirmed by quantitation of HOECHST-stained condensed nuclei (Figure 3c). Cardiac myocytes were cultured for 72 hours under hypoxia without medium change, with or without the addition of sufficient buffer and alkali to neutralize the pH every 12 hours. These cells were fixed and double stained with HOECHST 33342 and MF-20 (Figure 1). Examples are shown in Figure 3d. Hypoxic, pH-neutralized myocytes exhibited some degree of myofilament deterioration, but cross-striations were still apparent, and most of the nuclei were normal (compare with Figure 1e). In the field shown, 25 nuclei were scored normal and 3 were apoptotic; 1 nucleus was localized to a nonmyocyte.
Induction of apoptosis by extracellular lactic acid. The results described here demonstrate that low [pH]o is critical for the induction of apoptosis by hypoxia in this model. They do not show that acidosis can activate apoptosis independently of other factors that may accumulate or disappear from the extracellular medium during exposure to hypoxia. To test the effect of acidosis directly, we added lactic acid to fresh cardiac myocyte cultures at similar concentrations to those seen in conditioned medium after 72 hours of hypoxia (48), and adjusted the pH to 6.8 with phosphoric acid. These cultures were exposed to aerobic or hypoxic conditions as before. As shown in Figure 4, the addition of acid induced DNA laddering in both aerobic and hypoxic cardiac myocytes. The fragmentation induced by exogenous acid was not as pronounced as that caused by 24 hours of conditioned medium (Figure 2). This could be due to a number of factors, including the site of generation of the acid or additional proapoptotic components in the conditioned medium. However, there was more DNA fragmentation in the acidified aerobic cultures than in either the aerobic or hypoxic controls, even though the hypoxia control cultures achieved the same final pH. These results confirm that extracellular acidosis can induce significant apoptosis independently of hypoxia in cardiac myocytes.
Exogenous lactic acid induces apoptosis. Lactic acid was added to culture medium to a final concentration of 16.5 mM, and the pH was adjusted to 6.8 by adding phosphoric acid. The medium was added to fresh cultures of cardiac myocytes, and the cultures were incubated under air or hypoxia as indicated. DNA fragmentation was determined as described in Methods. The initial pH in both aerobic and hypoxic cultures was 6.8; the aerobic cultures maintained this pH, but the pH of hypoxic cultures decreased further to a final pH of 5.8.
Hypoxia-acidosis–mediated apoptosis is independent of changes in p53. The results described here demonstrate that extracellular acidosis, and possibly other metabolites, induces apoptosis of cardiac myocytes independently of hypoxia. Because p53 has been implicated in numerous pathways of apoptosis in other systems, as well as in cardiac myocytes subjected to stretch, angiotensin II, and hypoxia (42–44), we asked whether p53 was involved in initiating the signaling pathways mediated by hypoxia-acidosis. Four assays for p53 activity are shown in Figure 5, a–d. Figure 5a shows typical Western blots of proteins from cardiac myocytes and mouse embryo fibroblasts (MEFs) subjected to increasing periods of hypoxia (as in Figure 1, a and b). The p53 levels in the myocytes were very low at all times and did not change after short- or long-term exposure to hypoxia, with or without metabolic buildup. Infection with a p53 adenovirus caused a large accumulation of p53 (Figure 5a, third panel). As a positive control, p53 was measured in MEFs exposed to hypoxia (Figure 5a, bottom panel). In agreement with a previous report (43), p53 protein was elevated approximately 4-fold in these cells after 12 hours.
Hypoxia-acidosis–mediated apoptosis is independent of p53. (a) Cardiac myocytes were exposed to hypoxia as described in Methods. Plates were harvested at the indicated times, and proteins were extracted. For the 36-hour time points in a, the culture medium was changed every 12 hours (no metabolite buildup) or there was no medium change (metabolite buildup). In the bottom panel, proteins were extracted from MEFs as described in Methods. Western blots and probes are described in Methods. (b) Nuclear extracts were prepared from aerobic and (unfed) hypoxic cultures and from cultures infected for 48 hours with an adenoviral vector expressing p53 as described in Methods. Competition lanes include self (p53) and an oligonucleotide with the hypoxia-inducible factor-1 consensus binding site (HRE) (73). All probes and procedures are described in Methods. Equal amounts of protein (8 μg) were loaded in each lane. (c) Cardiac myocytes were exposed to hypoxia (without medium change) for the indicated times; protein was extracted and analyzed by Western blot using anti-Bax, anti-Bak, and anti-actin antibodies, described in Methods. (d) Cardiac myocytes were transfected with the indicated plasmid using calcium phosphate as described in Methods. Transfected cultures were incubated under aerobic or hypoxic conditions for 24, 48, or 72 hours (without medium change) as indicated. For viral infections, transfected cultures were inoculated with wild-type p53 at 5 PFU/cell plus 5 PFU of a virus expressing LacZ (Ad-p53), or with 5 PFU/cell of wild-type p53 plus 5 PFU of dominant-negative p53 (Ad-p53DN; contains a Cys-135→Ser mutation; ref. 74) as described in Methods. (e) Cardiac myocytes were isolated from wild-type or p53 knockout neonatal mice as described in Methods. The myocytes were cultured and exposed to hypoxia under the same conditions described in Figure 1b.
Changes in the activity of p53 may occur independently of net synthesis, e.g., through phosphorylation or nuclear translocation (59, 60). To determine whether there were changes in the activity of nuclear p53, DNA binding was measured by electrophoretic mobility shifts using a p53 binding site oligonucleotide and nuclear extracts from aerobic and hypoxic cardiac myocytes (as described in Methods). Previous reports have demonstrated the weak but specific nature of p53 binding by this assay (42, 43). The arrow in Figure 5b indicates the p53-specific binding complex. This band was competed by excess cold probe (lane 5), but not by excess of a non–p53 binding site probe (HRE, lane 6), and it was not present when the labeled probe contained a p53 binding site mutation (lanes 7–10). There was no change in the specific p53 binding complex between aerobic and hypoxic nuclear extracts. In agreement with a previous report (44), infection with a p53 adenovirus caused an increase of p53 complex binding (lane 4).
Changes in the expression of p53 target genes Bax and Bcl2 have been implicated in apoptosis caused by pressure overload, angiotensin II, and stretch (42). Increased expression of Bcl2 and Fas proteins and no change of Bax were reported in ischemic myocardial tissue (61). Western blot assays were used to determine whether chronic hypoxia caused changes of Bax, Bcl2, or Bak. As shown in Figure 5c, exposure of myocytes to hypoxia for 24 or 48 hours caused no apparent changes in Bak or Bax; Bcl-2 protein levels were too low to detect, consistent with a previous report (62).
To assay for changes in function of p53, we measured the effects of chronic hypoxia on the expression of a transfected p21 promoter (43, 44). As shown in Figure 5d, there was no significant effect of hypoxia on the expression of luciferase from either wild-type or mutant p21 promoters (in p21-M, the p53 binding sites of the wild-type p21 promoter are deleted). The slight trend toward increased p21 promoter activity after 48 hours of hypoxia (1.2 ± 0.3–fold), although differing from the trends of the α-MHC and RSV promoters at this time point, was not significant when compared with the p21 aerobic control or the p21-M promoter at 48 hours of hypoxia (0.9 ± 0.15). As a positive control, p21-Luc–transfected cells were infected with a p53 adenovirus (p53-Ad); in this case, expression of the p21 promoter was induced by about 3-fold. When the p21-Luc–transfected cultures were infected with equal amounts (5 PFU/cell) of Ad-p53 and an adenoviral vector containing a dominant-negative mutant p53 (Ad-p53DN), the induction was quenched (Figure 5d, lane 10). Expression of the hypoxia-responsive vector pα-MHC–HRE-Luc (lanes 11 and 12) confirmed that the culture conditions used here were sufficient to activate hypoxia-regulated genes, whereas there was no effect on the expression of the RSV promoter (47, 63).
Finally, we detected no change in endogenous p53 or p21 transcripts by Northern blots of RNA from cardiac myocytes exposed to hypoxia or hypoxia-acidosis for 6, 12, 24, or 36 hours, compared with aerobic controls (data not shown).
These results indicate that p53 activity in cardiac myocytes does not change during the exposure of these cells to chronic hypoxia and suggest that the ensuing apoptosis is independent of changes in p53 activity. To confirm this, cardiac myocytes were isolated from wild-type and p53 knockout mice and were subjected to chronic hypoxia-acidosis (without medium change) as described in Figure 1b. As shown in Figure 5e, although the levels of apoptosis were lower than previously seen with rat cardiac myocytes, there was clear genomic DNA fragmentation in both wild-type and knockout mouse cells; the arrows in the figure indicate fragments of about 200-bp increments, highly characteristic of apoptotic genomic DNA (2). No fragmentation was detected in the control (aerobic) samples. There appeared to be slightly more DNA loaded in the knockout lanes, and more intense fragmentation; densitometric scanning indicated approximately equivalent ratios of fragmented/intact DNA in the 2 samples (n = 2; data not shown). The reason for lower levels of apoptosis in mouse versus rat cardiac myocytes is probably related to the slightly decreased contractility and slower rate of acid production in the mouse cultures (data not shown). In other experiments, rat myocytes treated with Ad-p53DN, as described in Figure 5d, displayed identical DNA fragmentation as the controls after exposure to chronic hypoxia-acidosis (data not shown). Together, these results strongly suggest that hypoxia-acidosis–mediated apoptosis of cardiac myocytes is independent of p53 activity.
Reoxygenation of hypoxic cardiac myocytes induces apoptosis. The results reported here show that chronic hypoxia alone does not cause apoptosis of cardiac myocytes. Our previous results indicated that chronic hypoxia caused loss of glutathione and mediated a stress response that included the induction of c-Jun NH2-terminal kinase (JNK) activity when the cultures were reoxygenated (64). These conditions mimic the changes in oxygen tension that occur during ischemia and reperfusion. We therefore asked whether reoxygenation of hypoxic cultures also induced apoptosis. These results are shown in Figure 6, a and b. There was no evidence of apoptosis after 24 hours of hypoxia, in agreement with the results in Figure 1. However, DNA laddering was apparent 8–12 hours after reoxygenation. Maximal DNA fragmentation occurred 16–24 hours after reoxygenation and then declined to baseline levels. Figure 6b shows representative fields of HOECHST-stained nuclei. Arrows in the bottom left panel indicate examples of condensed nuclei that were scored positive. In this field, 12 nuclei were scored condensed and 15 were normal. Figure 6c shows the quantitation of condensed nuclei under the different conditions, determined as described in Figure 1; approximately 30% of the cells became apoptotic at the peak time of 16–24 hours after reoxygenation.
Induction of apoptosis by hypoxia-reoxygenation. Cultures of cardiac myocytes were exposed to hypoxia for 24 hours and then reoxygenated by replacing the medium with fresh aerobic medium and incubating in air (21% O2/5% CO2). Plates were harvested at the indicated times and analyzed for DNA fragmentation (a) or nuclear condensation (b and c) as described in Methods. HOECHST staining and the quantitation of condensed nuclei were as described in Figure 1. Typical fields of HOECHST-stained cardiac myocytes (b) were photographed at ×400. The 48-hour hypoxic incubation (c) was subjected to medium replacement and did not become acidic.
Reoxygenation-mediated apoptosis is independent of changes in p53. To determine the role of p53 or its associated genes in reoxygenation-mediated apoptosis, we measured protein levels of p53, Bak, Bax, and Bcl-2 at intervals during exposure of cells to hypoxia-reoxygenation. These results are shown in Figure 7. As with hypoxia alone, there were no major changes in the levels of p53, Bak, or Bax after hypoxia-reoxygenation. Bcl-2 was not detectable. The slight increase in p53 seen at the 24-hour hypoxia time point was not reproduced in 2 other experiments in which there was a slight decrease and no change (data not shown).
To confirm these results, we asked whether hypoxia-reoxygenation induced DNA fragmentation of cardiac myocytes in the absence of p53. Myocytes were isolated from p53-null homozygous knockout mice and their wild-type littermates and were subjected to hypoxia-reoxygenation (Figure 6). The results are shown in Figure 8. DNA internucleosomal cleavage peaked earlier and was at least equivalent in cardiac myocytes from p53–/– versus p53+/+ animals. This confirms that apoptosis mediated by either hypoxia-reoxygenation or hypoxia-acidosis does not require p53.
Induction of apoptosis in cardiac myocytes from p53–/– knockout mice. Cardiac myocytes were isolated from 1- to 2-day-old wild-type (a) or p53-nullizygous (b) mice as described in Methods. Cell culture and treatments were exactly as described in Figure 6.
Ischemia with reperfusion causes equivalent apoptosis in wild-type and p53 knockout mouse hearts. As one approach to determine whether the results obtained from the in vitro model reflected pathological changes in the intact organ, we measured DNA fragmentation from mouse hearts subjected to ischemia and reperfusion. Adult hearts from wild-type and p53 knockout mice were perfused by the Langendorff technique as described in Methods. Previous studies demonstrated that DNA fragmentation induced by 24 hours of permanent occlusion of the coronary artery (ischemia alone) occurred at equivalent levels in wild-type and p53 knockout mice (46). Apoptosis induced by ischemia and reperfusion of Langendorff-perfused mouse hearts has not been reported previously. Figure 9 compares nucleosomal ladders from the 2 groups. There was clear genomic DNA fragmentation in both wild-type and p53 knockout cells subjected to 20 minutes of ischemia and 3 hours of reperfusion (Figure 9, panels 2 and 4, respectively). DNA fragmentation in the knockout hearts was at least equivalent to that in the wild-type. No fragmentation was detected in the aerobic samples (controls, 3-hour aerobic perfusion; Figure 9, all panels) or in samples from hearts subjected to ischemia alone for 20 minutes to 3 hours (Figure 9, panels 1 and 3); a faint smear of degraded DNA was sometimes evident in samples subjected to extended ischemia, but no distinct ladders were seen. In the ischemia-reperfused samples (Figure 9, panel 2), DNA ladders appeared only after more than 2 hours of reperfusion, and the degree of fragmentation was maximal after 3–4 hours of reperfusion. Apoptosis in the ischemia-reperfused hearts of both wild-type and p53 knockout mice was confirmed by TUNEL assay, using an ApopTag kit (Intergen Co., Purchase, New York, USA) in which TUNEL-positive cells colocalized with cardiac myocytes (data not shown). These results show that ischemia with reperfusion, but not ischemia alone, induces equivalent DNA fragmentation in Langendorff-perfused hearts from wild-type and p53–/– knockout mice.
Nucleosomal fragmentation in genomic DNA from ischemia-reperfused mouse hearts. Hearts were perfused on a Langendorff apparatus as described in Methods. Left ventricle DNA was isolated from wild-type and p53–/– knockout mice subjected to ischemia alone or to ischemia and reperfusion. Controls were perfused with aerobic perfusion buffer (95% O2/5% CO2) for 3 hours. Nucleosomal ladders are apparent from the ischemia-reperfused hearts of both genotypes.
Hypoxia, acidosis, and reoxygenation do not induce DNA fragmentation in nonmuscle fibroblasts. Finally, in results not shown, confluent cultures of nonmuscle fibroblasts were exposed to the same conditions of hypoxia, acidosis, and reoxygenation as described earlier here for cardiac myocytes, and were analyzed for DNA fragmentation. There was no evidence of DNA fragmentation in these cells under any condition, even under the most extreme conditions of hypoxia with acidosis. This indicates that the responses described here are at least partially specific for cardiac myocytes; it also suggests that the apoptosis observed and quantitated in this report is probably exclusively from cardiac myocytes.