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Am J Physiol Heart Circ Physiol 287: H1466-H1475, 2004. First published May 20, 2004; doi:10.1152/ajpheart.00874.2003
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Na+/Ca2+ exchanger plays a key role in inducing apoptosis after hypoxia in cultured guinea pig ventricular myocytes

B. N. Eigel, H. Gursahani, and R. W. Hadley

Department of Molecular and Biomedical Pharmacology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536-0298

Submitted 11 September 2003 ; accepted in final form 13 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Altered Na+/Ca2+ exchanger (NCX) protein expression or activity is thought to contribute to various aspects of cardiac pathology. In guinea pig ventricular myocytes, NCX-mediated Ca2+ entry is almost entirely responsible for Ca2+ overload during hypoxia-reoxygenation. Because Ca2+ overload is a common initiator of apoptosis, the purpose of this study was to test the hypotheses that NCX activity is critically involved in initiating apoptosis after hypoxia-reoxygenation and that hypoxia-reoxygenation-induced apoptosis can be modulated by changes in NCX protein expression or activity. An NCX antisense oligonucleotide was used to reduce NCX protein expression in cultured adult guinea pig ventricular myocytes. Caspase-3 activation and cytochrome c release were used as markers of apoptosis. Hypoxia-reoxygenation-induced apoptosis was significantly decreased in antisense-treated myocytes compared with untreated control or nonsense-treated myocytes. Pretreatment of cultured myocytes for 24 h with either endothelin-1 or phenylephrine was found to increase both NCX protein expression and evoked NCX activity as well as enhance hypoxia-reoxygenation-induced apoptosis. Control experiments demonstrated that endothelin-1 and phenylephrine did not induce apoptosis on their own nor did they enhance the apoptotic response in a model of Ca2+-dependent, NCX-independent apoptosis. Additional control experiments demonstrated that the NCX antisense oligonucleotide did not alter the apoptotic response of myocytes to either H2O2 or isoproterenol. Taken together, these data suggest that the NCX has a critical and specific role in the initiation of apoptosis after hypoxia-reoxygenation in guinea pig myocytes and that hypoxia-reoxygenation-induced apoptosis is quite sensitive to changes in NCX activity.

heart; endothelin; phenylephrine; ischemia

THE CARDIAC NA+/CA2+ EXCHANGER (NCX) has been shown to be important in cardiac pathology. The NCX contributes to an increase in the cytosolic Ca2+ concentration ([Ca2+]i) in various species after ischemia or hypoxia (15, 26, 38) and is the predominant mechanism of Ca2+ overload after hypoxia-reoxygenation in isolated guinea pig ventricular myocytes (8). Elevated [Ca2+]i is believed to be a central mediator of myocardial injury and cell death (31) and has been shown to lead to arrhythmogenesis and necrosis (33, 40).

Several studies have led to the increasing appreciation that changes in cardiac NCX expression or activity could contribute to cardiac pathophysiology. NCX expression or activity is often increased in cardiac hypertrophy or heart failure in humans and animal models (2, 39, 42). A number of pharmacological agents known to induce cardiac hypertrophy including endothelin-1 (ET-1), ANG II, and {alpha}-adrenergic agonists can stimulate cardiac NCX activity (1, 20, 50). NCX mRNA, protein expression, and current were all found to be increased in a rat model of heart failure (32). In another study (23), pressure overload-induced heart failure in cats was found to increase NCX mRNA levels. Aortic banding-induced hypertrophy in mice was also found to increase NCX mRNA and protein expression (48). Increased gene expression of the cardiac NCX has also been reported in end-stage human heart failure (19, 42), which suggests that NCX upregulation could be a compensatory mechanism, although this could lead to deleterious side effects such as arrhythmogenesis (42). Consistent with this idea is a report that in rabbit heart failure, NCX overexpression enhanced the magnitude of the arrhythmogenic transient inward current (33). Finally, overexpression of cardiac NCX in mice has been reported to enhance ischemic injury (Ref. 4, but see Ref. 17).

An aspect of cardiac pathophysiology that has recently drawn increased interest is a possible role of apoptosis in hypoxic or ischemic injury (36, 49, 51). Apoptotic signaling pathways are of course complex, and several factors commonly implicated in hypoxia or ischemia (e.g., elevated [Ca2+]i or reactive oxygen species) are also involved in the initiation of apoptosis (10). Consideration of these intricate signaling pathways raises the issue of the relative importance of NCX-mediated Ca2+ overload to the triggering of apoptosis in hypoxic myocytes.

We previously studied [Ca2+]i overload in isolated adult guinea pig ventricular myocytes in response to hypoxia-reoxygenation, which is a key component of ischemia-reperfusion. Other studies have shown that [Ca2+]i is elevated in various models of ischemia (31, 40) and that the NCX could be responsible for this rise in [Ca2+]i (16, 45). In our model, an elevated cytosolic Na+ concentration precedes the [Ca2+]i overload that occurs during reoxygenation (7, 34), and this overload is clearly due to Ca2+ influx through reverse-mode NCX activity. The key evidence in support of this comes from adult cultured guinea pig ventricular myocytes treated for 5–6 days with an antisense oligonucleotide directed to the cardiac NCX1 start site (8). This antisense oligonucleotide treatment almost completely suppressed sarcolemmal NCX protein expression, evoked reverse-mode NCX activity, and the increase in [Ca2+]i after hypoxia-reoxygenation.

The purpose of this study was to follow up our previous results by testing the hypothesis that the NCX could serve as a novel link between hypoxia and apoptosis in adult guinea pig ventricular myocytes. Experiments were carried out in cultured adult myocytes in a long-lasting preparation that allowed us to perform longer term experiments involving the induction of apoptosis and the modulation of NCX protein expression and function. An NCX antisense oligonucleotide was used to decrease NCX protein expression, whereas ET-1 or phenylephrine (PE) was used to increase NCX activity. These studies led us to the conclusions that 1) the cardiac NCX has a prominent role in initiating apoptosis in adult guinea pig ventricular myocytes under the specific conditions of hypoxia-reoxygenation, and 2) alterations in NCX activity can be an important mechanism for modulating hypoxia-reoxygenation-inducedapoptosis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Experiments were conducted on cultured adult guinea pig ventricular myocytes. Female Hartley guinea pigs were anesthetized with an intraperitoneal injection of pentobarbital sodium before hearts were excised. This method of euthanasia was approved by the University of Kentucky Institutional Animal Care and Use Committee.

Cultured adult myocytes were prepared using common sterile techniques (8, 25). Isolated myocytes were suspended in serum-free medium 199 that was supplemented with (in mmol/l) 25 HEPES, 5 creatine, 2 L-carnitine, 5 taurine, and 10–4 insulin; also included were 0.2% BSA, 100 IU of penicillin, and 100 µg/ml streptomycin. MatTek glass-bottom, 35-mm microwell dishes were coated with 6 µg of Cell-Tak cell adhesive (BD Biosciences; Bedford, MA). Myocytes were plated at a density of 104 cells/cm2 and allowed to attach for 4 h after which the medium was removed and replaced with fresh medium. Myocytes were kept under sterile conditions in a 5% CO2 incubator at 37°C.

Hypoxia-reoxygenation protocol. A modular incubator chamber (Vangard International; Neptune, NJ) was used to make cultured myocytes hypoxic. Media 199 was first removed and replaced with modified minimum essential media (supplemented as described above for medium 199), which is low in sodium bicarbonate. The dishes of cultured myocytes were then placed in the chamber along with a dish that contained the oxygen indicator methylene blue. The chamber was then closed, and 100% nitrogen was flushed through the chamber until the methylene blue indicator was nearly colorless (~45 min). The nitrogen line was then removed and the chamber was sealed and placed in an incubator at 37°C for 6 h. At the end of 6 h, the culture dishes were removed from the chamber and placed in an incubator chamber with 5% CO2 at 37°C for 1 h of reoxygenation.

Measurement of apoptosis. Apoptosis was measured using both a caspase-3 activity assay and image analysis of cytochrome c release from mitochondria. We chose these methods because they allowed us to classify individual myocytes as apoptotic or nonapoptotic and thus facilitated quantitative measurements and statistical analyses of apoptosis in multiple treatment groups through cell counting. We evaluated other methods, particularly DNA laddering, but found that the caspase-3 and cytochrome c methods produced the most quantitative results.

PhiPhiLux, a fluorescent caspase-3 substrate, was used to assay myocytes for caspase-3 activity (OncoImmunin; Gaithersburg, MD). PhiPhiLux is a peptide that contains a caspase-3 recognition sequence (DEVD) tagged with a fluorescein isothiocyanate fluorophore. The uncleaved peptide is cell permeable and weakly fluorescent, whereas the cleaved peptide is cell impermeant and strongly fluorescent. PhiPhiLux (2.5 µmol/l) was added to each myocyte culture dish at the start of reoxygenation and allowed to incubate for 1 h at 37°C. Myocytes were gently washed once and then imaged using a laser-scanning confocal microscope (RCM 8000, Nikon; Melville, NY) and the 488-nm line of an Ar laser. Myocytes were classified as apoptotic or nonapoptotic based on the presence or absence of a fluorescent signal. Typical examples are shown in Fig. 1, A and B. Figure 1A shows bright-field images of a normoxic cardiac myocyte and a myocyte that was exposed to our standard hypoxia-reoxygenation protocol. Figure 1B shows the corresponding fluorescence images of caspase-3 activity. The hypoxic-reoxygenated myocyte is highly fluorescent, which suggests activation of caspase-3 and apoptosis, whereas the normoxic myocyte is essentially nonfluorescent.



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  		Fig. 1. Imaging of nonapoptotic and apoptotic cultured adult guinea pig ventricular myocytes. A: bright-field images of representative myocytes obtained under normoxic conditions (left) or after 6 h of hypoxia and 1 h of reoxygenation (right). Bar, 8 µm. B: corresponding images of the same myocytes as in A, demonstrating caspase-3 activation measured using a fluorescent caspase-3 substrate. C: cytochrome c distribution in representative myocytes imaged using immunocytochemistry and a monoclonal anti-cytochrome c antibody. A normoxic myocyte (left) and a hypoxic-reoxygenated myocyte (right) are shown. Bar, 20 µm.

 
Cytochrome c immunocytochemistry was carried out after fixation of myocytes in 4% paraformaldehyde and permeabilization with 0.1% Triton X-100. A monoclonal mouse anti-cytochrome c antibody (clone 6H2.B4, Research Diagnostics) was used in conjunction with a goat anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology) to visualize cytochrome c immunofluorescence. Confocal fluorescence images were obtained using the 488-nm line of an Ar laser. Myocytes were classified as nonapoptotic by the presence of a punctuate cytochrome c distribution, whereas apoptotic myocytes were classified by the presence of a nonpunctuate cytochrome c distribution. Figure 1C shows examples of both a normoxic myocyte with a nonapoptotic, punctuate cytochrome c distribution and a hypoxic-reoxygenated myocyte with an apoptotic, nonpunctuate distribution.

NCX immunocytochemistry. Myocytes were fixed and permeabilized as described above. A monoclonal mouse anti-NCX antibody (Research Diagnostics), which was developed using purified canine cardiac NCX as an antigen, was used in conjunction with a goat anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology) to visualize NCX immunofluorescence. Confocal fluorescence images were acquired as described for cytochrome c immunocytochemistry. NCX immunofluorescence in these images was quantified by masking all regions of the myocyte except the plasmalemma and then measuring the mean fluorescence intensity using MetaMorph software (Universal Imaging).

Fluorescence measurements of [Ca2+]i. We measured [Ca2+]i in myocytes loaded with the fluorescent indicator indo-1-acetoxymethyl ester (2.5 µmol/l for 20 min at 22°C). Images were acquired on the confocal microscope using excitation wavelengths of 351–364 nm emitted by an Ar laser. Dual-emission images at wavelengths longer or shorter than 445 nm were acquired for indo-1. Standard ratiometric analysis procedures were applied to the indo-1 images as previously described (5, 34). The [Ca2+]i values are reported as relative changes in the indo-1 fluorescence ratio: an increase in [Ca2+]i is seen as an increase in the fluorescence ratio.

Oligonucleotides. Oligonucleotides were synthesized at the University of Kentucky Macromolecular Structure Analysis Facility or at Integrated DNA Technologies (Coralville, IA). An antisense oligonucleotide (5'-TCGCAGCATGTTGTACAATG-3') was targeted to a region around the start codon of the cardiac guinea pig NCX1 (–11 to +9). A second oligonucleotide (nonsense; 5'-TCTCGAACGTGTTCAAGATG-3') was used to control for any nonspecific or toxic effect of the antisense oligonucleotide. Both oligonucleotides had eight phosphorothioate-modified nucleotides (shown in bold). Cultured myocytes were treated with antisense (2 µmol/l), nonsense (2 µmol/l), or no oligonucleotide as appropriate. Fresh oligonucleotides and medium 199 were added every 48 h. Myocytes were maintained in culture for 5–7 days.

Cell counting and statistical analysis. Cell counts for the apoptosis measurements were made by classifying all nonhypercontracted myocytes in defined regions of the culture dish as apoptotic or nonapoptotic based on caspase-3 activation or cytochrome c release. The number of cells (n) for each dish ranged from 50–75 myocytes, and the baseline (control) apoptosis values in individual culture dishes ranged from 25–44%. Because the essentially permanent Cell Tak adhesive prevents release of dead or dying myocytes into the media, these procedures were needed to account for necrotic (hypercontracted) myocytes or myocytes that became apoptotic during the often long preexperimental incubations.

Differences between means were analyzed using one-way or two-way ANOVA (general linear model) as appropriate. Student-Newman-Keuls multiple-comparison testing was used for post hoc significance testing between appropriate groups. Variance is described as the standard errors of the mean.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NCX1 antisense oligonucleotide protects cardiac myocytes from hypoxia-reoxygenation-induced apoptosis. Adult guinea pig ventricular myocytes were maintained in culture for 5–7 days in the presence of either a nonsense (2 µmol/l), an antisense (2 µmol/l), or no oligonucleotide. This antisense treatment protocol has been previously shown to almost completely suppress both sarcolemmal NCX protein expression and NCX-mediated [Ca2+]i accumulation during hypoxia-reoxygenation (8). The quiescent myocytes were subjected to 6 h of hypoxia and 1 h of reoxygenation to evaluate a potential role of the NCX in the initiation of hypoxia-reoxygenation-induced apoptosis. Caspase-3 activation and cytochrome c release from mitochondria were used as markers of apoptosis in parallel experiments as shown in Fig. 1.

Figure 2A shows a statistical analysis of caspase-3 measurements made after 6 h of hypoxia and 1 h of reoxygenation. Hypoxia alone induced a significant increase in the number of apoptotic myocytes (159 ± 5% relative to normoxic control myocytes). A nonsense oligonucleotide failed to protect the myocytes from hypoxia-reoxygenation-induced apoptosis (156 ± 5%). However, hypoxia-reoxygenation-induced apoptosis was significantly reduced in antisense-treated myocytes (109 ± 4%) compared with either hypoxia alone or nonsense-treated myocytes. Figure 2B shows a similar analysis of myocytes that demonstrates release of cytochrome c from mitochondria after 6 h of hypoxia and 1 h of reoxygenation. Hypoxia alone induced a significant increase in the number of apoptotic myocytes (168 ± 3% relative to normoxic control myocytes). A nonsense oligonucleotide failed to protect the myocytes from hypoxia-reoxygenation-induced apoptosis (166 ± 3%). However, hypoxia-reoxygenation-induced apoptosis was significantly reduced in antisense-treated myocytes (118 ± 4%) compared with either hypoxia alone or nonsense-treated myocytes.



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  		Fig. 2. Pretreatment with a Na+/Ca2+ exchanger (NCX) antisense oligonucleotide but not a nonsense oligonucleotide reduced apoptosis in guinea pig ventricular myocytes subjected to hypoxia-reoxygenation. A: caspase-3 activation was measured after 6 h of hypoxia and 1 h of reoxygenation. B: cytochrome c release was measured under the same conditions as in A. C: caspase-3 activation was measured after myocytes were subjected to 12 h of hypoxia followed by 1 h of reoxygenation. D: cytochrome c release was measured under the same conditions as in C. All myocytes were cultured for 5–7 days with either no, nonsense (2 µmol/l), or antisense (2 µmol/l) oligonucleotides. *P < 0.05 vs. hypoxic control. Each bar represents the mean ± SE of either six (for 6-h data) or three (for 12-h data) 35-mm culture dishes. Data were normalized to untreated normoxic control myocytes.

 
Figure 2, C and D, shows the measurement of caspase-3 activation and cytochrome c release in myocytes that were subjected to 12 h of hypoxia followed by 1 h of reoxygenation. As expected, the number of apoptotic myocytes was somewhat higher than after 6 h of hypoxia. Pretreatment with the NCX antisense oligonucleotide provided significant protection against hypoxia-reoxygenation-induced apoptosis, but the degree of protection was less extensive. These results provide strong evidence that the NCX has a prominent role in initiating apoptosis after a period of hypoxia-reoxygenation in adult guinea pig ventricular myocytes. A comparison of the 6- and 12-h hypoxia data in Fig. 2 suggests that the role of the NCX in hypoxia-reoxygenation-induced apoptosis is most prominent at moderate durations of hypoxia and that NCX-independent mechanisms may be expected to predominate at more prolonged durations of hypoxia.

Decreasing NCX protein expression does not affect H2O2- or isoproterenol-induced apoptosis. Our next experiments were designed to answer the question of whether the protective effect of the NCX antisense oligonucleotide was specific to hypoxia-reoxygenation (when NCX mediates Ca2+ influx) or whether antisense pretreatment had a more generalized, nonspecific protective effect against cardiac apoptosis. Two agents that can be used to induce cardiac myocyte apoptosis in the absence of hypoxia are H2O2 and isoproterenol (Iso; Refs. 41, 44). We treated guinea pig ventricular myocytes with various concentrations of either H2O2 (20–100 µmol/l) or Iso (1–10 µmol/l) for 24 h and found that 20 µmol/l H2O2 and 5 µmol/l Iso reliably induced caspase-3 activation and cytochrome c release. We used these experimental conditions for our next experiments.

Figure 3 shows a statistical analysis of the effects of the NCX antisense oligonucleotide on H2O2- and Iso-induced apoptosis. Myocytes were again maintained in culture for 5–7 days in the presence of either a nonsense (2 µmol/l), an NCX antisense (2 µmol/l), or no oligonucleotide. Myocytes were then treated for 24 h with either 20 µmol/l H2O2 or 5 µmol/l Iso, which produced a sizeable increase in the number of cells undergoing apoptosis as shown in Fig. 3. Pretreatment of myocytes with the antisense oligonucleotide did not significantly affect H2O2- or Iso-induced caspase-3 activation (Fig. 3A) or cytochrome c release (Fig. 3B). In separate experiments, we also examined the effects of Iso pretreatment on NCX protein expression on the plasma membrane. Iso had no significant effect (99.1 ± 2.2%, compared with control cells).



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  		Fig. 3. Pretreatment with the NCX antisense oligonucleotide does not prevent H2O2- or isoproterenol (Iso)-mediated apoptosis. A: caspase-3 activation was measured by a fluorescent caspase-3 substrate. B: cytochrome c release was measured by immunocytochemistry. Myocytes were treated for 5–7 days with no, nonsense (2 µmol/l), or antisense (2 µmol/l) oligonucleotides and then treated for 24 h with either 20 µmol/l H2O2 or 5 µmol/l Iso. There was no significant difference between antisense-treated myocytes and nonsense-treated or control myocytes. Each bar represents the mean ± SE of six 35-mm culture dishes.

 
ET-1 and PE pretreatments increase NCX activity. The results shown in Fig. 2 demonstrated that the NCX had an important role in initiating apoptosis in cardiac myocytes subjected to hypoxia-reoxygenation. We next wanted to evaluate the additional hypothesis of whether relevant stimuli such as activation of cardiac endothelin or {alpha}-adrenergic receptors could enhance hypoxia-reoxygenation-induced apoptosis, and whether these effects could be attributed to altered NCX expression or activity. Previous reports (1, 13, 50) have shown that NCX activity or expression could be regulated by ET-1 or PE. We first carried out NCX immunocytochemistry experiments to define experimental conditions where ET-1 and PE were most likely to stimulate NCX activity. Figure 4A shows images of NCX immunofluorescence in a control myocyte and in a myocyte treated with 10 nmol/l ET-1 for 24 h. Plasmalemmal NCX immunofluorescence was moderately enhanced in the ET-1-treated myocyte. Figure 4B shows the effect of 2–10 nmol/l ET-1 and 20–100 µmol/l PE on the mean plasmalemmal NCX immunofluorescence intensity. It can be seen that 10 nmol/l ET-1 (130 ± 5%) and 100 µmol/l PE (120 ± 4%) had the largest effects on NCX protein expression compared with untreated controls (100%). These experimental conditions were used in all subsequent ET-1 and Iso experiments.



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  		Fig. 4. Pretreatment with endothelin-1 (ET-1) or phenylephrine (PE) significantly increased NCX activity. A: NCX protein expression was imaged using immunocytochemistry and confocal microscopy in a control cultured myocyte and in a myocyte treated with 10 nmol/l ET-1 for 24 h. B: plasmalemmal NCX immunofluorescence was quantified in control myocytes and in myocytes treated with ET-1 or PE for 24 h. *P < 0.05 vs. untreated control; n = 8 myocytes. C: reverse-mode NCX activity was evaluated in indo-1-acetoxymethyl ester-loaded myocytes by measuring changes in cytosolic Ca2+ concentration ([Ca2+]i) during exposure first to K+-free and then to Na+-free extracellular solutions. An increase in [Ca2+]i was seen as an elevated indo-1 fluorescence ratio. Some myocytes were pretreated for 24 h with either 10 nmol/l ET-1 or 100 µmol/l PE before being transferred to drug-free media for 1 h. Additional groups of myocytes were treated with both ET-1 and 100 nmol/l BQ-123 or PE and 1 µmol/l prazosin (PRAZ). *P < 0.05 vs. control value at the same time point; n = 6 myocytes.

 
Figure 4C summarizes the results of experiments done to confirm that 24 h of pretreatment with either 10 nmol/l ET-1 or 100 µmol/l PE was sufficient to significantly increase NCX activity in guinea pig ventricular myocytes. Cultured myocytes were first pretreated with either ET-1, PE, or no drug (control) as described (see NCX1 antisense oligonucleotide protects cardiac myocytes from hypoxia-reoxygenation-induced apoptosis) and were then subjected to an assay of evoked reverse-mode NCX activity 1 h after the drugs were washed off. The assay consisted of first loading the myocytes with the fluorescent indicator indo-1 so that [Ca2+]i could be measured. The myocytes were then exposed to a K+-free media, which is well known to inhibit the Na+-K+-ATPase and elevate the cytosolic Na+ concentration. After 10 min, this solution was replaced with a Na+-free media, which stimulated a rapid rise in [Ca2+]i. This increase in [Ca2+]i is due to NCX-mediated Ca2+ influx (reverse-mode activity) and is absent in myocytes pretreated with the NCX antisense oligonucleotide (8). Figure 4C shows that either ET-1 or PE pretreatment of the cardiac myocytes significantly enhanced reverse-mode NCX activity in this assay. The stimulatory effect of ET-1 or PE on the NCX could be prevented by addition of either the ETA receptor antagonist BQ-123 or the {alpha}-adrenergic receptor antagonist prazosin at the time of addition of either ET-1 or PE.

Pretreatment with ET-1 or PE enhances hypoxia-reoxygenation-induced apoptosis. Because we had established that both ET-1 and PE could enhance NCX protein expression and activity, the next step in evaluating our hypothesis was to examine the effects of ET-1 and PE on apoptosis in cardiac myocytes. Guinea pig ventricular myocytes were cultured for 5–7 days with either 2 µmol/l nonsense, 2 µmol/l antisense, or no oligonucleotide and were also pretreated for 24 h with either 10 nmol/l ET-1 or 100 µmol/l PE. The drugs were washed off 1 h before the myocytes were exposed to 6 h of hypoxia and 1 h of reoxygenation. Apoptosis was assessed by measuring either caspase-3 activation (Fig. 5A) or cytochrome c release (Fig. 5B) at the end of 1 h of reoxygenation. Figure 5A shows that in myocytes treated with no oligonucleotide, pretreatment with ET-1 or PE significantly increased caspase-3 activation compared with hypoxic controls, which demonstrates that ET-1 and PE can significantly increase hypoxia-reoxygenation-induced apoptosis (*P < 0.05). Additional statistical analysis revealed that caspase-3 activation in myocytes pretreated with both the antisense oligonucleotide and ET-1 or PE was significantly lower than in myocytes pretreated with either ET-1 or PE alone. Note that hypoxic control myocytes were not directly compared with myocytes pretreated with both drug (ET-1 or PE) and oligonucleotides due to the presence of more than one variable. Figure 5B shows similar results using cytochrome c release as a marker of apoptosis. Also note that the effects of ET-1 and PE on hypoxia-reoxygenation-induced apoptosis could be prevented by BQ-123 or prazosin. The caspase-3-activation and cytochrome c-release data suggest first that ET-1 and PE can significantly enhance hypoxia-reoxygenation-induced apoptosis, and second, that this effect is dependent on NCX protein expression.



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  		Fig. 5. Pretreatment with either ET-1 or PE significantly increased NCX-dependent apoptosis in myocytes subjected to hypoxia-reoxygenation. Myocytes were cultured for 5–7 days in the presence of either 2 µmol/l nonsense, 2 µmol/l NCX antisense, or no oligonucleotide. Some groups of myocytes were then pretreated for 24 h with either 10 nmol/l ET-1 (with or without 10 nmol/l BQ-123) or 100 µmol/l PE (with or without 1 µmol/l prazosin). Drugs were washed off 1 h before myocytes were exposed to 6 h of hypoxia and 1 h of reoxygenation. A: caspase-3 activation was measured using a fluorescent caspase-3 substrate. B: cytochrome c release was measured using immunocytochemistry. *P < 0.05 vs. hypoxic control; {dagger}P < 0.05 vs. myocytes treated with no oligonucleotides. Each bar represents the mean ± SE of six 35-mm culture dishes.

 
NCX oligonucleotides, ET-1, and PE do not increase apoptosis during normoxia. The results shown in Figs. 4 and 5 suggest that pretreatments that modulate NCX protein expression and activity can also modulate apoptosis after hypoxia-reoxygenation. Our final experiments examined two alternative explanations for our data: first, that pretreatment of myocytes with either ET-1 or PE could itself induce apoptosis; and second, that ET-1 or PE pretreatment could cause a general enhancement of the myocyte response to Ca2+-dependent apoptosis (not just when Ca2+ enters the myocyte through the NCX).

To determine whether the pretreatments alone might affect apoptosis, we investigated whether the oligonucleotides ET-1 or PE could alter caspase-3 activation or cytochrome c release under normoxic conditions. Myocytes were maintained in culture for 5–7 days and either treated with a nonsense (2 µmol/l), an NCX antisense (2 µmol/l), or no oligonucleotide or else were treated for 24 h with either 10 nmol/l ET-1 or 100 µmol/l PE. Myocyte apoptosis was measured 1 h after the end of the pretreatment period using caspase-3 activation and cytochrome c release as markers of apoptosis.

Figure 6 shows that pretreatment with the nonsense or antisense oligonucleotides ET-1 or PE did not significantly alter either caspase-3 activation (Fig. 6A) or cytochrome c release (Fig. 6B) compared with untreated cultured control myocytes. These data suggest that the pretreatments themselves in the absence of the hypoxia-reoxygenation protocol do not have a significant effect on apoptosis in this preparation.



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  		Fig. 6. Oligonucleotide, ET-1, or PE pretreatment alone does not induce apoptosis in normoxic myocytes. A: caspase-3 activation was measured using a fluorescent caspase-3 substrate. B: cytochrome c release was measured by immunocytochemistry. Myocytes exposed to oligonucleotides alone were maintained in culture for 5–7 days and treated with either 2 µmol/l nonsense or 2 µmol/l NCX antisense oligonucleotide. Myocytes exposed to drugs alone were treated for 24 h with either 10 nmol/l ET-1 or 100 µmol/l PE. Each bar represents the mean ± SE of four to six 35-mm culture dishes.

 
Figure 7 shows the results of experiments designed to test whether ET-1 or PE could enhance Ca2+-dependent but NCX-independent apoptosis in guinea pig ventricular myocytes. Myocytes were cultured for 5 days and then pretreated with either 10 nmol/l ET-1, 100 µM PE, or no drug. The drugs were then washed off, and the myocytes were exposed to the Ca2+ ionophore 4-Br-A23187 (1 µmol/l) for 2 h. The purpose of the ionophore was to induce increases in [Ca2+]i and Ca2+-dependent apoptosis through a mechanism that did not depend on NCX activity. The 4-Br-A23187 alone induced an increase in the indo-1 fluorescence ratio to 141 ± 7% (n = 8) of the baseline value. Figure 7, A and B, summarizes the results of studies of caspase-3 activation and cytochrome c release under these conditions. As can be seen by the increased caspase-3 activation and cytochrome c release, 4-Br-A23187 induced apoptosis in these myocytes. Figure 7 also demonstrates that pretreatment of the myocytes with either ET-1 or PE did not affect either measurement. These results are consistent with the idea that ET-1- and PE-enhanced hypoxia-reoxygenation induced apoptosis (as illustrated in Fig. 5) through an NCX-dependent mechanism rather than by modulating later signaling events associated with Ca2+-dependent apoptosis.



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  		Fig. 7. PE and ET-1 had no effect on apoptosis in a cultured myocyte model of Ca2+-dependent, NCX-independent apoptosis. Myocytes were cultured for five days before being exposed to either 10 nmol/l ET-1, 100 µmol/l PE, or no drugs for 24 h. Drugs were then washed off before myocytes were exposed to 1 µmol/l 4-Br-A23187 for 2 h. A: caspase-3 activation was measured using a fluorescent caspase-3 substrate. B: cytochrome c release was measured by immunocytochemistry. Each bar represents the mean ± SE of six 35-mm culture dishes.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A critical role for NCX in mediating hypoxia-reoxygenation-induced apoptosis in guinea pig ventricular myocytes. In our previous studies, we established that the NCX mediates the rise in [Ca2+]i that is seen immediately upon reoxygenation of hypoxic guinea pig ventricular myocytes (8), an effect that has been linked to the necrotic death of cardiac myocytes (31, 40). In contrast, there has been little evidence to implicate the NCX in apoptotic myocyte death. After it was first recognized in ischemic rabbit myocytes (14), cardiac apoptosis has since been described and characterized in a number of species and ischemia models (11, 22, 49, 51). However, the contribution of apoptosis to overall cell death after acute ischemia-reperfusion remains ill defined (9, 12, 36). Because elevated [Ca2+]i can be a common mechanism for inducing apoptosis (10), the aim of this study was to evaluate whether the cardiac NCX is a crucial link between hypoxia and the induction of apoptosis.

In this study, we tested the hypothesis that the NCX has a critical role in the initiation of apoptosis in guinea pig ventricular myocytes after a period of 6 h of hypoxia and 1 h of reoxygenation. This protocol was designed to produce a moderate level of metabolic stress in our cellular model. Apoptosis was measured independently by two separate assays: activation of caspase-3 and release of cytochrome c from the mitochondria into the cytosol. It should be noted that the latter measure is a hallmark of the mitochondrial apoptosis signaling pathway, which is commonly involved in Ca2+-dependent apoptosis (10). Our most important result is that pretreatment of cultured adult guinea pig ventricular myocytes with an antisense oligonucleotide, which has been previously shown to almost completely suppress NCX protein expression and Ca2+ accumulation during reoxygenation (8), inhibits hypoxia-reoxygenation-induced apoptosis. This result provides strong support for our hypothesis and suggests a potentially new pathophysiological role for the NCX. We suggest that the NCX can make a strong contribution to the initiation of apoptosis only under conditions that promote Ca2+ influx through the NCX, which is often referred to as "reverse-mode" activity. This could happen in hypoxic guinea pig ventricular myocytes due to the accumulation of sodium in the cytosol (7, 34) but may be less relevant to many other proapoptotic situations such as exposure of normoxic myocytes to H2O2 or Iso.

Previous studies of Iso- or H2O2-induced apoptosis are consistent with these observations. The mechanisms involved in Iso-induced apoptosis are still unclear (27) but have been proposed to involve such signaling pathways as calcineurin (37) or Ca2+/calmodulin-dependent protein kinase II activation (52). Several signaling pathways have been suggested to be involved in H2O2-induced apoptosis including direct promotion of cytochrome c release (30) and NF-{kappa}B activation (47). It was also recently reported that 100 µmol/l H2O2 stimulated the Na+/H+ exchanger in cultured neonatal rat cardiomyocytes, which led to elevated cytosolic Na+ and Ca2+ levels and apoptosis (46). However, our data (shown in Fig. 3) make it clear that under our specific experimental conditions (20 µmol/l H2O2 and cultured adult guinea pig ventricular myocytes), the participation of the NCX is not required for H2O2-induced apoptosis.

NCX in cardiac myocytes is a target for modulation of hypoxia-reoxygenation-induced apoptosis. It has become increasingly clear that a number of factors can enhance cardiac NCX protein expression and/or activity and that this enhancement in NCX activity has significant pathophysiological consequences including altered contractile function and arrhythmogenesis (2, 42). In this study, we used relatively low concentrations of ET-1 and PE to stimulate NCX expression and activity in adult cultured myocytes. Our results (see Fig. 5) indicate that an increased sensitivity to hypoxia-reoxygenation-induced apoptosis is an unfavorable consequence of enhanced activity. A number of the factors that have been established to enhance NCX mRNA or protein levels are implicated in heart failure. NCX message or protein levels have been demonstrated to be elevated in several animal models of heart failure (18, 23, 32, 48) and in human heart-failure patients (43). ET-1, ANG II, and catecholamine levels are also elevated in heart failure (21), and it has been established that activation of endothelin, angiotensin, and {alpha}-adrenergic receptors can stimulate NCX activity (1, 20, 50). In many of these studies, it is likely that much of this stimulatory effect occurs through modulation of preexisting NCX proteins. For example, ET-1, at least at high concentrations (100 nmol/l), has been reported to induce the phosphorylation of NCX1 (20). However, because we observed that a stimulatory effect of ET-1 or PE was persistent for >1 h after washout of these drugs (see Fig. 4C) and that these drugs increased NCX protein expression (see Fig. 4B), some part of the stimulatory effect of these agonists may be mediated through a moderate increase in the number of NCX1 transporters found on the sarcolemma. In agreement with this, PE was previously shown to upregulate NCX1 gene translation (3) and to enhance NCX message and protein expression in rat cardiac myocytes (35).

Limitations of this study. We would like to point out two limitations to our conclusion that the NCX has a critical role in the initiation of hypoxia-reoxygenation-induced apoptosis. The first is suggested by the data in Fig. 2, C and D, where a particularly prolonged hypoxic period induced substantial apoptosis even in myocytes that had been pretreated with the NCX antisense oligonucleotide. This may suggest the existence of alternative apoptotic signaling pathways that do not involve the NCX. This is not surprising, considering the numerous relevant signaling pathways that are undoubtedly activated by the broad stress of hypoxia. Nevertheless, it is clear that in this model, NCX-triggered apoptosis is the predominant mechanism early on. The second limitation is that our studies were necessarily carried out in cultured hypoxic myocytes, whereas apoptotic signaling in intact, ischemic myocardium is undoubtedly more complex. Although hypoxia is a key component of myocardial ischemia, the model is admittedly simpler than in vivo models. Hypoxia-reoxygenation is meant to mimic key features of ischemia-reperfusion, in particular, the metabolic and oxidative stress, but obvious differences exist between this model and true ischemia. Nevertheless, our observations of hypoxic myocytes are likely relevant to intact myocardium, as NCX-dependent Ca2+ accumulation is also thought to occur in ischemic myocardium (40).

Some methodological limitations of this study should also be mentioned. Differences in myocyte morphology or function should be expected between freshly isolated and cultured myocytes including the gradual loss of transverse tubules (24). However, in this study, cultured myocytes were maintained in a serum-free environment, which slows potential changes over time. Previous electrophysiological studies have shown that NCX activity, when normalized to cell capacitance, remains unchanged in adult guinea pig ventricular myocytes for at least 4 days in culture (29). In addition, we previously reported (8) that the magnitude of evoked reverse-mode NCX activity as measured using the assay shown in Fig. 4C is not significantly different in cultured or freshly isolated guinea pig ventricular myocytes.

ET-1 and PE were used to test the hypothesis that hypoxia-reoxygenation-induced apoptosis would be sensitive to drugs that modulated the level of NCX protein expression and activity. ET-1 and PE of course have other known effects on cardiac myocytes besides their actions on the NCX. An alternative approach would have been to overexpress the NCX1 in these experiments. However, this approach has its own potential limitations for these apoptosis studies, as NCX overexpression might induce endoplasmic stress and modulate apoptosis through two undesired mechanisms: the unfolded protein response (28) or unloading of intracellular Ca2+ stores (6). We therefore used ET-1 and PE to ensure a more moderate enhancement of NCX activity and expression. There are several observations that support our conclusion that the effects of ET-1 and PE on apoptosis in this model are mediated through altered NCX activity. It should first be pointed out that ET-1 and PE were always used as pretreatments; the drugs were washed off the myocytes for at least 1 h before we began either our NCX or hypoxia experiments. Any acute, nonpersistent actions of the drugs should therefore have had a negligible effect on our measurements. Even more important are our observations that the effects of ET-1 and PE to enhance hypoxia-reoxygenation-induced apoptosis were dependent on NCX expression (see Fig. 5) and that the drugs had no effect on myocyte apoptosis either in the absence of hypoxia (see Fig. 6) or in a model of Ca2+-dependent, NCX-independent apoptosis (see Fig. 7).

In conclusion, this study provides strong evidence that the NCX can initiate apoptosis in adult guinea pig myocytes after a period of hypoxia-reoxygenation and that hypoxia-reoxygenation-induced apoptosis is sensitive to altered levels of Ca2+ entry through reverse-mode NCX transport.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grant HL-56910 and National Institute on Aging Grant AG-10836 (to R. W. Hadley).


   ACKNOWLEDGMENTS
 
Present addresses: B. Eigel, American Heart Association, National Center, 7272 Greenville Ave., Dallas, TX 75231; and H. Gursahani, Internal Medicine and Cardiovascular Medicine, TB-172, Bioletti Way, University of California, Davis, CA 95616.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Hadley, Dept. of Molecular and Biomedical Pharmacology, Univ. of Kentucky, College of Medicine, MS-371 UKMC, Lexington, KY 40536-0298 (E-mail: rhadley{at}uky.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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