Cardiac ischemia/reperfusion (I/R) injury can result in many heart diseases such as myocardial infarction and heart failure. There are many mechanisms involved in myocardial I/R injury including excessive production of reactive oxygen species, disrupting the integrity of the mitochondrial intermembrane space, mitochondrial calcium overload, and efflux of cytochrome C and other proapoptotic factors (1, 2). The consequences of all these lead to cardiomyocyte apoptosis.
Apoptosis is controlled by the Bcl-2 family members. P53 upregulated modulator of apoptosis (PUMA) is a member of Bcl-2 family. It is originally identified as p53 downstream target gene and is a critical mediator of p53-dependent apoptosis (3, 4). Deletion of PUMA in cells inhibits the apoptotic response to p53, DNA-damaging agents, and hypoxia. Knockout of PUMA in mice almost completely suppresses p53-dependent apoptosis. Furthermore, PUMA deficiency protects cells from diverse apoptotic stimuli such as cytokine withdrawal or exposure to the glucocorticoid dexamethasone, the kinase inhibitor staurosporine, and phorbol esters, indicating that PUMA can also mediate p53-independent death pathways (5). Therefore, PUMA is a powerful proapoptotic factor.
It has been reported that PUMA is involved in cardiomyocyte apoptosis. For example, endoplasmic reticulum (ER) stress promotes PUMA expression and apoptosis in neonatal cardiomyocytes. Inhibition of PUMA expression by adenoviral delivery of shRNA decreases the cultured cardiomyocyte apoptosis induced by ER stress, and cardiomyocytes from wild-type and PUMA+/− mice but not from PUMA−/− mice are sensitive to ER stress and undergo apoptosis (6). Transfection of normal cardiomyocytes with adenoviruses expressing a wild-type PUMA protein leads to apoptosis. Furthermore, hearts of PUMA−/− mice are significantly resistant to I/R injury. Infarct sizes in the PUMA−/− hearts were greatly reduced. And heart function recovery was significantly improved in PUMA−/− mice upon I/R (7). It seems that PUMA is an essential mediator in cardiomyocyte apoptosis induced by I/R injury. However, the mechanisms by which PUMA mediates I/R-induced cardiomyocyte apoptosis are largely unknown.
Our present work used the in vitro model to elucidate the effects of PUMA on hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis as well as the underlying mechanisms. Our data showed that H/R led to an elevation in PUMA mRNA and protein levels in part by p53-dependent manner. Targeted deletion of PUMA attenuated H/R-induced cardiomyocyte apoptosis. Furthermore, we found that PUMA mediated H/R-induced cardiomyocyte apoptosis by triggering mitochondrial apoptotic pathway.
MATERIALS AND METHODS
One-day-old male Sprague-Dawley rats were used for cardiomyocyte culture. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1996). The research was approved by the Animal Research Councils and the Ethical Council, Health Center, Peking University, China.
Neonatal rat cardiomyocyte cultures and treatment
Neonatal rat cardiomyocyte cultures were prepared as we previously described (8).
Cells were transfected with 100 nmol/L siGENOME SMARTpool reagent specific for rat PUMA and nontargeting siRNA-control (Thermo Scientific Dharmacon) using DharmaFECT siRNA Transfection Reagents (Thermo Scientific Dharmacon, Lafayetta, Colo). Forty-eight hours after transfections, the culture dishes were placed into a hypoxic incubator with 95% N2-5% CO2 at 37°C. After 2-h hypoxia, the dishes were transferred to a normoxic incubator for reoxygenation.
Cell death assessment
Cell death was determined by trypan blue exclusion, and the numbers of trypan blue-positive and -negative cells were counted on a hemocytometer.
Flow cytometry analysis of cell apoptosis
Cells were labeled with annexin V and propidium iodide (PI) (Becton-Dickinson, Franklin Lakes, NJ) according to the manufacturer's instructions. Briefly, 1 × 105 cells were washed with cold phosphate-buffered saline, and resuspended with binding buffer, then transferred to a 5-mL tube. Annexin V and PI were added to the cell preparations and incubated for 25 min in the dark. Four hundred microliters of binding buffer was added to the tubes, and the samples were analyzed by flow cytometry.
Semiquantitative reverse transcriptase-polymerase chain reaction
RNA was isolated from cardiomyocytes using TRIzol reagent (TransGen Biotech) and processed according to the manufacturer's instructions. For quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), cDNAs were synthesized from 1 μg of total RNA with the Quantscript RT kit (TianGen Biotech, Beijing, China). Polymerase chain reaction was carried out using the following PUMA primers: 5-TGGGTGCACTGATGGAGATA-3 (forward) and 5-AACCTATGCAATGGGATGGA-3 (reverse). GAPDH served as a loading control.
Cells were lysed at 4°C in a lysis buffer (20 mM HEPES pH 7.7, 2.5 mM MgCl2, 0.1 mM EDTA, 20 mM β-glycerophosphate, 0.5 mM DTT, 0.1 mM sodium orthovanadate, 75 mM NaCl, 0.05% Triton X-100). For detection of cytosolic cytochrome C, the cytosolic fractions were prepared. Cells were washed twice with phosphate-buffered saline, and the pellet was suspended in 0.2 mL of buffer A (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.1 mM phenylmethanesulfonyl fluoride, 250 mM sucrose) containing a protease inhibitor cocktail. The cells were homogenized on ice using a tight-fitting Dounce homogenizer. The homogenates were centrifuged at 2,500 revolutions per minute (rpm) for 10 min at 4°C. The supernatants were centrifuged at 14,000 rpm for 25 min at 4°C to get the cytosolic fractions (supernatants). Samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore Corporation, Bedford, Mass). The blotted membranes were incubated with primary antibodies including anti-PUMA, anti-p53, and anti-cytochrome C antibodies, followed by a peroxidase-conjugated secondary antibody. Antigen-antibody complexes were visualized by enhanced chemiluminescence.
Immunoprecipitation was prepared as described by Li et al. (9). The samples were precleared with 5% (vol/vol) protein-G agarose (Roche) for overnight on a rocking platform. Specific antibodies were added and rocked for 1 h. Immunoprecipitates were captured with 5% (vol/vol) protein-G agarose overnight. The agarose beads were spun down and washed three times with wash buffer. The antigens were released and denatured by adding SDS sample buffer. Immunoblot analysis was performed as described above.
Detection of mitochondrial membrane potential
Mitochondrial membrane potential (Δψm) was detected using MitoProbe JC-1 Assay Kit (Invitrogen, Carlsbad, Calif). Cells in the various treatment groups were digested with trypsin and resuspended in warm phosphate-buffered saline at a concentration of 1 × 106 cells/mL. JC-1 was added into phosphate-buffered saline at the final concentration of 2 μM. Then, cells were incubated at 37°C 5% CO2 for 30 min. Afterward, cells were washed by warm phosphate-buffered saline and centrifuged. The supernatants were removed, and cell pellets were resuspended in 500 μL phosphate-buffered saline and immediately analyzed by flow cytometry with 488-nm excitation.
The results were expressed as the mean (SD) (n = 3). For the comparison between two groups, the Student t test was used. A one-way ANOVA was used for multiple comparisons. P < 0.05 was considered significant.
H/R-induced cardiomyocyte apoptosis requires PUMA
As shown in column 2 in Figure 1A, H/R increased cardiomyocyte death monitored by trypan blue exclusion (P < 0.001 compared with control). To confirm whether the cell death occurred by apoptosis, neonatal rat cardiomycytes were labeled with annexin V/PI and subsequently analyzed by flow cytometry. As shown in Figure 1, B and C, H/R induced cardiomyocyte apoptosis (P < 0.001 compared with control). Concomitantly, PUMA mRNA and its protein levels were upregulated in cardiomyocytes upon treatment with H/R (Fig. 1D, column 2). To quantify PUMA expression levels, we used Image-Pro Plus software to analyze the integrated optical density (IOD) of PUMA bands. As shown in column 2 in Figure 1, E and F, H/R increased the expression of PUMA (P < 0.001 vs. control). To explore whether there is a link between cell apoptosis and PUMA upregulation, we tested whether inhibition of endogenous PUMA can influence the cell fate upon treatment with H/R. Inhibition of PUMA was achieved by using its siRNA (siRNA-PUMA). The nontargeting siRNA (siRNA-control) was used as the control. siRNA-PUMA, but not siRNA-control, inhibited the expression of endogenous PUMA (Fig. 1, D-F, columns 3-4; P < 0.001 vs. H/R). Similarly, siRNA-PUMA, but not siRNA-control, prevented apoptosis induced by H/R (Fig. 1, A-C, columns 3-4). These data indicate that H/R-induced cardiomyocyte death requires PUMA.
H/R-induced PUMA expression is, in part, p53 dependent
To understand whether p53 is a necessary regulator of H/R-induced PUMA expression, we first analyzed the levels of p53 and PUMA proteins. As shown in Figure 2, A and B, p53 protein levels were upregulated in cardiomyocytes upon treatment with H/R. Concomitantly, PUMA protein levels were also increased in cardiomyocytes exposed to H/R (Fig. 2, A and C). Then, we detected cell apoptosis in response to H/R, respectively, by trypan blue exclusion and flow cytometry. Figure 2, D and E, shows that apoptosis was increased by H/R in parallel with the levels of p53 and PUMA protein. It seems that p53 is related to PUMA and apoptosis. To prove this issue, we treated cells with pifithrin-α, a p53 inhibitor. As shown in column 3 in Figure 3, A-C, pifithrin-α decreased the expression levels of PUMA mRNA and its protein (P < 0.01 compared with H/R). However, it is noted that although the levels of PUMA mRNA and its protein were decreased, there still remained an amount of PUMA levels in response to H/R (P < 0.01 compared with control). Similarly, we found that pifithrin-α decreased H/R-induced apoptosis (Fig. 3, D and E, column 3; P < 0.05), but the percentage of apoptosis in pifithrin-α-treated group is higher than that in the control group (P < 0.05 compared with control). These results indicate that H/R-induced PUMA expression is, in part, p53 dependent.
The decrease in the association of ARC with caspase 8 contributes to apoptosis under H/R condition
ARC under the physiological condition is associated with caspase 8. However, in the presence of PUMA, PUMA interacts with ARC, thereby releasing caspase 8 and inducing apoptosis. We asked whether H/R can influence the association of ARC with caspase 8 or PUMA. To answer this question, we used immunoprecipitation to detect the association of ARC with caspase 8 or PUMA under H/R condition. As shown in Figure 4, A-C, in the control group, the association levels of ARC with caspase 8 were high, and the associations of ARC with PUMA were weak. In contrast, PUMA, but not caspase 8, was strongly associated with ARC in cells undergoing H/R. However, when PUMA was suppressed using its siRNA (siRNA-PUMA) under H/R condition, caspase 8, but not PUMA, was strongly associated with ARC. Concomitantly, caspase 8 activation (Fig. 4D) and cell death (Fig. 1, A-C) could be increased while increasing the association levels of PUMA with ARC. Thus, the data suggest that the increase in the association of PUMA with ARC may substitute that of ARC with caspase 8.
PUMA is essential for the loss of mitochondrial membrane potential in cardiomyocytes upon treatment with H/R
To determine the apoptotic pathway of H/R mediated by PUMA, we measured the mitochondrial membrane potential (ΔΨm) by flow cytometry using the fluorescent probe JC-1. JC-1 is selectively taken up into mitochondria and thus is a reliable indicator of ΔΨm. In the nonapoptotic cells with intact mitochondria, JC-1 dye appears as a monomer in the cytosol and emits green fluorescence. While accumulating as aggregates in the mitochondria, it emits red fluorescence. Therefore, the nonapoptotic cells display high green and red fluorescence and appear in the upper-right region of the flow cytometric scatter plot. In contrast, in the apoptotic cells JC-1 cannot accumulate in the mitochondria, and it remains in monomer form in the cytosol. These cells display high green but low red fluorescence and appear in the lower right (LR) region of the scatter plot. As shown in Figure 5, H/R led to an increase in the cell percentages in the LR region (P < 0.01 compared with control). However, siRNA-PUMA that suppresses endogenous PUMA expression attenuated the cell percentages in the LR region (P < 0.01 compared with H/R). Taken together, these data indicate that PUMA is essential for the loss of mitochondrial membrane potential in cardiomyocytes upon treatment with H/R.
PUMA is involved in cytochrome C release and caspase 3 activation in cardiomyocytes exposed to H/R
During mitochondria-mediated apoptosis, several mitochondrial apoptotic proteins, such as cytochrome C, are released into the cytosol to facilitate the activation of caspase cascade. To investigate the role of PUMA in H/R-induced cytochrome C release and caspase 3 activation, we tested cytochrome C expression of cytosolic fractions and the activation of caspase 3 in cardiomyocytes transfected with siRNA-PUMA or siRNA-control under H/R condition. Hypoxia/reoxygenation caused the increase in cytochrome C expression and caspase 3 activity (P < 0.001, compared with control). In contrast, when the endogenous PUMA was inhibited, cytochrome C expression and caspase 3 activation induced by H/R were decreased (P < 0.001, compared with H/R) (Fig. 6). These results suggest that induction of cytochrome C release and the activation of caspase 3 by H/R require the participation of PUMA.
Our present work shows that H/R-induced apoptosis and the increase in PUMA expression in cardiomyocytes are accompanied by the decrease in mitochondrial membrane potential, the release of cytochrome C, and activation of caspases 3 and 8. However, these effects of H/R disappear upon PUMA knockdown. These data suggest that PUMA mediates cardiomyocyte apoptosis triggered by H/R through mitochondrial pathway.
Although several studies have indicated that PUMA contributes to cardiomyocyte apoptosis induced by I/R injury, the mechanism of the effect of PUMA on I/R-induced cardiomyocyte apoptosis has not been established. Hypoxia/reoxygenation initiates the mitochondrial apoptotic pathway by inducing the loss of mitochondrial membrane potential, which releases cytochrome C from mitochondria into cytoplasm. In the cytosol, cytochrome C interacts with apoptotic protease activation factor 1, which binds to and activates caspase 9 and, in turn, its downstream caspase 3, resulting in apoptosis (10). Our present work reveals that H/R not only induces the loss of mitochondrial membrane potential and stimulates cytochrome C release and caspase 3 activation, but also increases PUMA expression. Intriguingly, when endogenous PUMA is inhibited, the loss of mitochondrial membrane potential, cytochrome C release, and caspase 3 activation induced by H/R were attenuated. Our data indicate that PUMA appears to mediate H/R-induced cardiomyocyte apoptosis through mitochondrial pathway.
The interactions between proapoptotic and antiapoptotic members play an important role in apoptosis initiation. It has been suggested that PUMA can bind to ARC. ARC is the first antiapoptotic protein so far identified to be highly expressed in cardiac and skeletal muscle tissues (11). ARC was originally identified to be a caspase inhibiting protein. Under the physiological condition, ARC is associated with caspase 8 (9). However, in the presence of PUMA, PUMA interacts with ARC, thereby releasing caspase 8 and inducing apoptosis. In our present work, we observed that the association levels between ARC and PUMA were elevated, whereas the association levels between ARC and caspase 8 were decreased. Knockdown of endogenous PUMA resulted in an increase in the association levels between ARC and caspase 8. These results suggest that the interaction between ARC and caspase 8 can be interrupted by PUMA upon H/R. Such an interruption leads to the activation of caspase 8, thereby activating the apoptotic program.
PUMA is identified as a p53-inducible gene (3, 4). In I/R-induced cardiomyocyte death, the effect of p53 on PUMA activation is somewhat controversial. Toth et al. (7) found that PUMA-null mice are more resistant to I/R than p53-null mice, thereby supposing that I/R-induced apoptosis requires PUMA, but not p53, and other transcription factors other than p53 play a role in PUMA activation in cardiomyocytes. However, more studies prove that I/R increases p53 expression in cardiomyocytes, and p53 is implicated in I/R injury (12-14). In this study, we observed that the elevation of p53 induced by H/R is accompanied by the increase in PUMA. P53 inhibitor attenuates the increase in PUMA and cell death induced by H/R, thereby demonstrating that p53 plays a role in H/R-induced PUMA activation. It is notable that the induction of PUMA expression and cardiomyocyte death after H/R are not completely blocked in p53 inhibitor-treated cells. Therefore, It is possible that upregulation of PUMA after H/R may be also facilitated by other transcription factors including p73 (15), E2F1 (16), and FOXO3a (17). It is necessary to test this hypothesis in future studies.
In summary, we have demonstrated that PUMA has a role in triggering cardiomyocyte apoptosis through mitochondrial pathway. And the elevation of PUMA in H/R-induced cardiomyocyte apoptosis is, in part, p53 dependent. These data shed new lights on understanding the complex signaling cascades involved in the cardiomyocyte injury upon H/R.
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PUMA; apoptosis; hypoxia/reoxygenation; cardiomyocyte