Differential Regulation of Cardiomyocyte Survival and Hypertrophy by MDM2, an E3 Ubiquitin Ligase*

MDM2 is an E3 ubiquitin ligase that regulates the proteasomal degradation and activity of proteins involved in cell growth and apoptosis, including the tumor suppressors p53 and retinoblastoma and the transcription factor E2F1. Although the effect of several MDM2 targets on cardiomyocyte survival and hypertrophy has already been investigated, the role of MDM2 in these processes has not yet been established. We have, therefore, analyzed the effect of overexpression as well as inhibition of MDM2 on cardiac ischemia/reperfusion injury and hypertrophy. Here we show that isolated cardiac myocytes overexpressing MDM2 acquired resistance to hypoxia/reoxygenation-induced cell death. Conversely, inactivation of MDM2 by a peptide inhibitor resulted in elevated p53 levels and promoted hypoxia/reoxygenation-induced apoptosis. Consistent with this, decreased expression of MDM2 in a genetic mouse model was accompanied by reduced functional recovery of the left ventricles determined with the Langendorff ex vivo model of ischemia/reperfusion. In contrast to cell survival, cell hypertrophy induced by the α-agonists phenylephrine or endothelin-1 was inhibited by MDM2 overexpression. Collectively, our studies indicate that MDM2 promotes survival and attenuates hypertrophy of cardiac myocytes. This differential regulation of cell growth and cell survival is unique, because most other survival factors are prohypertrophic. MDM2, therefore, might be a potential therapeutic target to down-regulate both cell death and pathologic hypertrophy during remodeling upon cardiac infarction. In addition, our data also suggest that cancer treatments with MDM2 inhibitors to reactivate p53 may have adverse cardiac side effects by promoting cardiomyocyte death.

The murine double minute 2 (mdm2) 2 gene was originally discovered as one of three genes that was overexpressed in spontaneously transformed mouse BALB/c fibroblasts (1,2). Overexpression of the mdm2 gene product was shown to lead to transformation, which requires the binding of MDM2 to the tumor suppressor p53 (3). Through this protein-protein interaction, MDM2 inhibits the transcriptional activity of p53 (4). In addition, MDM2 also promotes the ubiquitination and proteasomal degradation of p53 by functioning as an E3 ubiquitin ligase (5,6). p53 exerts its tumor suppressor effect by transcriptionally activating many target genes, including p21 and PUMA/Noxa, thereby promoting growth arrest both at G 1 and G 2 phases of the cell cycle and programmed cell death (apoptosis), respectively (7). Another transcriptional target of p53 is MDM2 itself, providing an autoregulatory negative feedback loop with a significant role in regulating cell cycle progression and apoptosis (7,8).
The importance of the MDM2/p53 interaction has been convincingly demonstrated by in vivo studies. Mice lacking mdm2 are embryonic lethal and die before implantation as early as the blastocyst stage (9). This phenotype is completely rescued by concomitant deletion of p53 (10,11), suggesting that the embryo lethality was due to active p53. In recent studies a mouse model with a hypomorphic allele has been developed that expresses ϳ30% of the total MDM2 levels (12), providing an excellent tool to examine the effects of the loss of MDM2 on adult animals. These mice have decreased body weight and defects in hematopoiesis and are more sensitive to ␥-irradiation than normal mice (12). The susceptibility of the hearts from these mice to ischemia/reperfusion, however, has not yet been characterized.
In addition to transcriptional regulation, several proteins interact with MDM2, leading to its posttranslational modification and changes in its ability to interact with p53 (9). One of these upstream regulators is the protein serine/threonine kinase Akt. It phosphorylates MDM2 at two consensus sites that results in stabilization of MDM2 via decreased ubiquitination (13)(14)(15). Interestingly, Akt has been implicated in both hypertrophy and cell survival of cardiomyocytes (16 -22), suggesting that MDM2 may be one of the downstream mediators of Akt to regulate these processes in the heart.
Although MDM2 is a critical regulator of the tumor suppressor p53 and has several other well defined p53-independent downstream targets that are potential regulators of cell survival and hypertrophy (9,(23)(24)(25)(26), the role of MDM2 in these fundamental processes in cardiac cells is poorly defined. Overexpression of MDM2 has been reported upon a variety of different treatments of cardiac myocytes. These include adenoviral delivery of wild-type p53 (27), treatment with the Akt-activator insulin-like growth factor 1 (28,29), induction of DNA damage by the cytostatic drug doxorubicin (30), and ischemic preconditioning (31). Moreover, insulin-like growth factor 1 was suggested not only to induce MDM2 but also to down-regulate p53, leading to attenuation of the renin-angiotensin system and stretch-mediated apoptosis (28,29). The role of MDM2 in ischemia/reperfusion injury and hypertrophy, however, has not yet been investigated.
In the current study we, therefore, analyzed the effect of MDM2 on cell survival and hypertrophy by using isolated cardiac myocytes as well as a genetic mouse model with reduced expression of MDM2. To our knowledge, these experiments demonstrate for the first time that the endogenous MDM2 plays a role in protection against cell death of ischemic/reperfused cardiomyocytes and negatively regulates hypertrophy. MDM2, therefore, emerges as a novel regulator of cardiac cell survival and growth and potentially may serve as a target for cardiac gene therapy.

EXPERIMENTAL PROCEDURES
Preparation of Adenoviruses-MDM2 adenovirus was generated by using the Adeno-X Tet-Off gene expression system (Clontech Laboratories). First, a Tet-responsive expression cassette was made by cloning the mouse mdm2 cDNA (2) (GenBank TM accession number X58876) into the pTRE-Shuttle plasmid. Then it was amplified in Escherichia coli, and the expression cassette was excised from pTRE-Shuttle with PI-SceI and I-CeuI and ligated into pAdeno-X Viral DNA (the adenoviral genome). The recombinant pAdeno-X was selected upon transformation of E. coli, then purified and linearized with PacI digestion. This linearized recombinant vector was packaged into infectious adenoviral particles by transfecting human embryonic kidney 293 cells (Microbix Biosystems) using Lipofectamine 2000 (Invitrogen). Recombinant adenoviruses were finally harvested by lysing transfected cells.
In Vitro Hypoxia/Reoxygenation-Two days after isolation, neonatal rat cardiomyocytes were cultured in glucose-and serum-free Dulbecco's modified Eagle's medium under hypoxic conditions as described earlier, with some modifications (34) (Fig. 1). Hypoxia was achieved by incubating cells in a humidified environment at 37°C in a 3-gas incubator maintained at 5% CO 2 and 1% O 2 for 12 h or for 4 h when it was combined with MDM2 inhibitor treatments (Fig. 1). Subsequently, half of the samples were reoxygenated for 16 h in maintenance medium. Certain samples were pretreated overnight with MDM2 adenoviruses or with an MDM2 inhibitor (PNC-28; 100 g/ml; Calbiochem), which is a peptide mimicking the MDM2 binding domain of p53 and carries a penetratin sequence to help the inhibitor enter the cells (35). We have used this MDM2-binding peptide domain of p53 because the x-ray structure of its complex with the MDM2 protein has been elucidated (36). The PNC-28 peptide corresponds to the specific region of human p53 between residues 12-26 (PLSQETFSDLWKLL) that contacts MDM2 (36). These data indicate that PNC-28 specifically binds to MDM2. After treatment, the samples were processed for immunocytochemistry or Western blots.
In Vitro Hypertrophy-Hypertrophy was analyzed as previously described with minor modifications (37) (Fig. 1). After isolation, cardiac myocytes were cultured in a serum-free Dulbecco's modified Eagle's medium containing 0.5% Nutridoma (Roche Applied Science). Cells were infected with the indicated adenoviruses 24 h after initial plating. 36 h after infection, cells were stimulated with phenylephrine (PE, 100 M) or endothelin-1 (ET-1, 100 nM) for an additional 36 h. Subsequently, the samples were fixed in 3.7% formaldehyde and processed for immunocytochemistry. Alternatively, in the last 24 h of the experiment, cells were incubated with 1 Ci/ml [ 3 H]leucine (PerkinElmer Life Sciences) to measure protein synthesis. Radioactivity incorporated into the trichloroacetic acid-precipitable material was determined by a liquid scintillation counter.
Immunocytochemistry-Cardiac myocytes were grown on laminincoated glass coverslips. Apoptotic cells were detected by the terminal deoxynucleotidyltransferase-mediated UTP in situ nick end labeling (TUNEL) method (Roche Applied Science). Sections were co-stained with anti-MDM2 (Ab2, Calbiochem) or anti-sarcomeric actinin (Sigma) antibodies, and the nuclear stains TO-PRO-3 or Syto-16 (Molecular Probes). Cardiomyocyte hypertrophy was demonstrated by staining the cells with antibodies against sarcomeric actinin and atrial natriuretic factor (ANF) (Peninsula Laboratories) together with TO-PRO-3. Images were analyzed by confocal fluorescence microscopy (Bio-Rad).
Generation of a Hypomorphic mdm2 Mouse Line-Mendrysa et al. (12) have recently developed a mouse model with a hypomorphic mdm2 allele. The mice in this model system may carry different combinations of three alleles. Besides the fully active wild-type allele (mdm2 wt ), these include the partially active hypomorphic mdm2 puro , which contains a puromycin resistance cassette, and the inactive mdm2 del7-9 , in which exons 7-9 have been deleted by Cre recombination. As a result, the mdm2 wt/del7-9 and mdm2 puro/del7-9 mice exhibited decreased MDM2 expression (ϳ50 and 30% of total MDM2, respectively) throughout the body when compared with the levels expressed in mdm2 wt/wt and mdm2 wt/puro mice (12).
We obtained mouse breeding pairs from the National Cancer Institute Mouse Repository and mated them according to a protocol published earlier (12). Briefly, mdm2 wt/del7-9 (B6.Cg genetic background) and mdm2 wt/puro (129/SvEv genetic background) mice were crossed and gave birth to mdm2 wt/wt , mdm2 wt/puro , mdm2 wt/del7-9 , and mdm2 puro/del7-9 mice. These mice were genotyped using the previously published primers (12), and the MDM2 protein expression was confirmed by immunoblots using mouse heart lysates (see Fig. 4A). All animal protocols were approved by the Institutional Animal Care and Use Committee.
Langendorff Heart Perfusion-The heart perfusion protocol we published earlier for rats was adapted for mice using a system purchased from ADInstruments (39). Briefly, mice were anesthetized with pentobarbital (60 mg/kg intraperitoneal), and the hearts were rapidly excised and placed into ice-cold Krebs-Henseleit buffer. Hearts were retrograde perfused at a constant pressure (80 mm Hg) with a flow rate of 1-3 ml/min. The perfusion buffer was a modified phosphate-free Krebs-Henseleit buffer that contained 118 mM NaCl, 4.7 mM KCl, 2 mM CaCl 2 , 1.2 mM MgSO 4 , 25 mM NaHCO 3 , and 11 mM glucose. The perfusate was equilibrated at 37°C with 95% O 2 , 5% CO 2 through a glass oxygenator to achieve pH 7.4. Hearts were exposed to a 20-min base-line perfusion, then subjected to 15-min no-flow ischemia and subsequently reperfused for 120 min. Hearts were paced at 7 Hz through platinum wires placed on the epicardial surface of the right ventricle; however, the pacer was turned off during ischemia. To measure left ventricular function, a small custom-made polyvinyl chloride balloon was inserted into the left ventricle through the mitral valve and filled to achieve an end-diastolic pressure of 8 -12 mm Hg. Functional data of mouse hearts (left ventricular developed pressure, dP/dt max , and dP/dt min ) were monitored dur-ing the entire perfusion using a data acquisition system (Powerlab, ADInstruments). To determine the infarct size, 2-mm-thick slices of the ventricle were immersed into 1% triphenyltetrazolium chloride solution (TCI America) in phosphate buffer at 37°C for 10 min (18). To detect necrotic membrane damage, coronary effluent was collected during base-line perfusion and reperfusion for lactate dehydrogenase activity measurement (U. S. Biological). For histological analysis, hearts were collected, fixed overnight in 10% formalin buffered with phosphatebuffered saline, dehydrated in ethanol, and transferred to xylene and then into paraffin. Paraffin-embedded hearts were sectioned at 4 m and subsequently stained with hematoxylin and eosin. The cardiac myocyte cross-sectional area was determined with the Image J software at the same magnification in the midventricular sections of each heart. The myocyte cross-sectional area was measured per nucleus, and only myocytes that were cut in the same direction were included in the measurement.

MDM2 Overexpression Protects Cardiac Myocytes against Hypoxia/
Reoxygenation-induced Apoptosis-To investigate the potential prosurvival activity of MDM2 in cardiac myocytes, isolated rat neonatal cardiomyocytes were infected with recombinant adenoviruses expressing MDM2 in a tetracycline-regulated manner using the Tet-Off system (see Fig. 1 for treatment protocol). Under these circumstances a high level of MDM2 expression was achieved in the absence of tetracycline (ϪTet) in most cells, with a transduction efficiency of 90% or more (Fig.  2, A-C). This high level of MDM2 expression was accompanied with increased Akt activity ( Fig. 2A) and maintained at least for 72 h (not shown). In contrast, overexpression of MDM2 was completely eliminated by the addition of tetracycline (ϩTet) to the culture medium (Fig.  2, A-C). The ability of the overexpressed MDM2 to promote p53 degradation has also been demonstrated ( Fig. 2A).
In control cells without adenoviral infection or with MDM2 virus infection under ϩTet conditions, hypoxia resulted in ϳ50% apoptosis scored by TUNEL assay (Fig. 2, C-D). Most importantly, MDM2 overexpression reduced the rate of apoptosis elicited by hypoxia by approximately half, resulting in 23% TUNEL-positive nuclei (Fig. 2, C-D). Hypoxia/reoxygenation-induced apoptosis followed a similar pattern; however, the cells were more sensitive and more than 90% of them underwent apoptosis. This was partially prevented by overexpression of MDM2, allowing 43% of the cells to survive (Fig. 2, C-D). In both cases the protective effect was attributable to elevated levels of MDM2 because infection with an MDM2 virus in the presence of tetracycline (or treatment with Ad␤-gal ϩ AdtTA ϩ Tet) did not prevent either hypoxia-or hypoxia/reoxygenation-induced apoptosis (Fig. 2, C-D). The specificity of the TUNEL assay for apoptosis in this model was confirmed by examining nuclear morphology using the DNA stain TO-PRO-3 (Fig. 2C).
These data indicate that MDM2 attenuates cardiac myocyte apoptosis in response to hypoxia/reoxygenation injury. Because earlier studies reported that targeted deletion of p53 is not sufficient to block the apoptotic response of cardiomyocytes exposed to ischemia/reperfusion (40,41), our findings also suggest that the ectopically overexpressed MDM2 does not exclusively act on p53 but also suppresses p53-independent cell death pathways.
MDM2 Inhibition Exacerbates Hypoxia/Reoxygenation-induced Cardiomyocyte Apoptosis-Because adenoviral delivery of MDM2 facilitated the survival of hypoxic/reoxygenated cardiac myocytes, we asked whether endogenous MDM2 is required for cardiomyocyte survival. To this end, we have analyzed the effect of MDM2 inhibition on the viability of cardiomyocytes during hypoxia/reoxygenation. To block MDM2 activity, we applied a peptide inhibitor (PNC-28) described earlier that mimics the MDM2 binding domain of p53 (35). It is thought to exert its effect primarily by occupying the p53 binding pocket of MDM2, thereby leading to stabilization of p53, followed by proteolytic activation of caspase-3 and apoptosis.
We administered PNC-28 to cardiac myocytes exposed to normoxia, hypoxia, or hypoxia/reoxygenation (see Fig. 1 for the treatment protocol). In these experiments we decreased the duration of the hypoxic period from 12 to 4 h because of the enhanced sensitivity of cells to double treatments. When the MDM2 inhibitor was used under normoxic conditions, ϳ30% of the cells died by apoptosis as opposed to less than 5% death of the non-treated cells (Fig. 3, A-B). This was accompanied by only a small increase in p53 levels and caspase-3 cleavage, similar to that observed under hypoxia or hypoxia/reoxygenation without the MDM2 inhibitor (Fig. 3C). When PNC-28 was combined with hypoxia  FEBRUARY 10, 2006 • VOLUME 281 • NUMBER 6  A, cardiomyocytes were grown on coverslips and treated with an MDM2 inhibitor (PNC-28, 100 g/ml) or left untreated. After overnight pretreatment, cardiomyocytes were exposed to 4 h hypoxia with or without reoxygenation (16 h). Cells were then stained for apoptosis by TUNEL assay (red), and cardiac myocytes were identified by staining with anti-sarcomeric actinin (green). Images were taken by confocal fluorescent microscope at 100ϫ magnification. B, quantification of cardiomyocyte apoptosis. The apoptotic nuclei were scored on the basis of TUNEL positivity as described in A. Data are averaged from three experiments in 10 randomly selected fields (at least 800 cells) from each group. The error bars are S.E. of the mean. *, significant difference from untreated normoxic sample (p Ͻ 0.01). #, significant difference from untreated hypoxic sample (p Ͻ 0.01). @, significant difference from untreated hypoxic/reoxygenated sample (p Ͻ 0.01). Hx, hypoxia. C, immunoblot analysis of MDM2, p53, and cleaved caspase-3 levels. Cardiomyocytes were grown in culture dishes and treated as described in A. Actin is shown as loading control. The results are representative of at least three similar experiments.

MDM2 Promotes Cardiomyocyte Survival but Blocks Hypertrophy
control. B, for immunocytochemistry, the nuclei were stained with Syto-16 (green), and overexpression of MDM2 was detected by an MDM2 antibody (red). The results are representative of at least three similar experiments. C, cardiomyocytes were grown on coverslips and infected overnight with MDM2 adenovirus (AdMDM2) and AdtTA viruses in the presence or absence of tetracycline. Cardiomyocytes were then exposed to hypoxia for 12 h with or without reoxygenation (16 h). Cells were stained for apoptosis by TUNEL assay (green) and for MDM2 (red), and nuclei were identified by staining with TO-PRO-3 (blue). Images were taken by confocal fluorescent microscope at 400ϫ magnification. D, quantification of cardiomyocyte apoptosis. The apoptotic nuclei were scored on the basis of TUNEL positivity as described in C. Data are averaged from three experiments in 10 randomly selected fields (at least 200 cells) from each group. The error bars are S.E. of the mean. *, significant difference from normoxic samples (p Ͻ 0.01). #, significant difference from the corresponding MDM2 ϩ Tet samples (p Ͻ 0.01). . Hypomorphic MDM2 expression increases the sensitivity of hearts to ischemia/reperfusion. Hearts from wild-type (n ϭ 9), mdm2 wt/puro (n ϭ 8), mdm2 wt/del7-9 (n ϭ 9), and mdm2 puro/del7-9 mice (n ϭ 8) were exposed to 15-min ischemia and 120-min reperfusion according to the Langendorff method. A, detection of MDM2 and p21 in hearts with different mdm2 genotypes by immunoblotting. Actin is shown as the loading control. The results are representative of three similar experiments. B, post-ischemic recovery of left ventricular developed pressure after 30, 60, 90, and 120 min of reperfusion compared with the base-line values. C, post-ischemic recovery of ϩdP/dt max and ϪdP/dt min after 120 min or hypoxia/reoxygenation, the two treatments potentiated each other, leading to apoptosis in ϳ90 and 95%, respectively (Fig. 3, A-B), and a stronger increase in p53 and cleaved caspase-3 (Fig. 3C). In addition, we detected similar levels of MDM2 expression in all samples except for a decrease upon hypoxia/reoxygenation, probably because of the cleavage of MDM2 by caspases (42).
Collectively, these data indicate that blocking MDM2 activity in cardiac myocytes can exacerbate hypoxia/reoxygenation injury. Our results imply that the endogenous MDM2 at least partially controls p53 in cardiac myocytes under both normoxic and hypoxic/reoxygenated conditions and promotes cardiomyocyte survival.

Impaired Tolerance of Hearts with Reduced MDM2 Expression to Ischemia/Reperfusion-Because inhibition of MDM2 activity sensitizes cardiac myocytes to hypoxia/reoxygenation-induced cell death in vitro,
we hypothesized that the endogenous MDM2 might be required for preserving the functional performance and cardiomyocyte viability of intact hearts challenged by ischemia/reperfusion. To test this hypothesis in adult animals, we utilized a transgenic mouse model with reduced MDM2 expression (see "Experimental Procedures"). We have chosen this hypomorphic mdm2 system because mdm2 knock-out mice are embryonic lethal, and generation of viable littermates without functional mdm2 gene is only feasible with simultaneous p53 gene deletion (9 -11). By circumventing the limitations of double-knockout studies, the hypomorphic MDM2 mouse model allowed us to assess the consequences of diminished MDM2 function in the presence of p53 in adult animals.
First, we determined the expression of MDM2 in the hearts of mice having different genotypes by immunoblots. In mdm2 wt/del7-9 mice, expression of MDM2 was reduced about 50% relative to mdm2 wt/wt and mdm2 wt/puro mice (Fig. 4A), consistent with 50% reduction in gene dosage. The level of MDM2 protein in hearts from mdm2 puro/del7-9 mice was further reduced to ϳ30% of the level of wild-type hearts (Fig. 4A). These results are consistent with measurements in other tissues, such as spleen, thymus, brain, liver, and kidney (12). In accordance with earlier studies (33, 43), we did not detect p53 protein in the mouse heart tissue lysates by Western blot. However, we measured induced p21 protein levels in the hypomorphic hearts, indicating increased p53 activity in hearts expressing less MDM2 (Fig. 4A), which is in line with previous reports (12). It was shown earlier that a decrease in MDM2 expression led to reduction of the body weight as well as the weight of several organs (12). As expected, we found that in mice expressing decreased levels of MDM2, the heart weight was also smaller ( Table 1).
Next we analyzed the functional performance of intact hearts using the Langendorff method. The isolated hearts were exposed to 15-min ischemia followed by reperfusion. Most importantly, hearts with decreased MDM2 expression (mdm2 wt/del7-9 and mdm2 puro/del7-9 ) showed impaired post-ischemic functional recovery compared with their wild-type littermates (mdm2 wt/wt and mdm2 wt/puro ) (Fig. 4, B-C). Although wild-type hearts retained 60 -70% of their base-line left ventricular developed pressure after 2 h of post-ischemic reperfusion, we observed a gradual loss of post-ischemic function as the expression of MDM2 diminished (Fig. 4B). The left ventricular developed pressure recovered only to 55 Ϯ 7% in mdm2 wt/del7-9 hearts and to 40 Ϯ 10% in mdm2 puro/del7-9 hearts (Fig. 4B). Similarly, the values of dP/dt max and dP/dt min were also decreased in animals with hypomorphic alleles as opposed to wild-type mice (Fig. 4C). However, no significant differences occurred in the base-line perfusion parameters of animals regardless of their genotype (Table 1).
We also measured the extent of the infarcted area by triphenyltetrazolium chloride staining in histological sections and measured lactate dehydrogenase release in the coronary effluent. Although the mdm2 wt/wt and mdm2 wt/puro hearts suffered about 30% infarction upon ischemia/reperfusion, we found 1.5-and 1.8-fold increased infarct size in mdm2 wt/del7-9 and mdm2 puro/del7-9 hearts, respectively (Fig. 4D). Consistent with the triphenyltetrazolium chloride assay, the release of lactate dehydrogenase into the perfusion fluid was also significantly elevated in mdm2 wt/del7-9 and mdm2 puro/del7-9 as opposed to mdm2 wt/wt and mdm2 wt/puro hearts (Fig. 4E). These findings establish MDM2 as an intrinsic prosurvival mediator of the heart under conditions of ischemia/reperfusion. MDM2 Overexpression Inhibits Cardiomyocyte Hypertrophy-Because many survival pathways also trigger cardiac hypertrophy (44,45), we examined whether MDM2 overexpression affects cardiomyocyte growth. To this end, we overexpressed MDM2 with the Tet-dependent adenoviral system demonstrated in Fig. 2, A-C, stimulated cardiac myocytes with PE or ET-1 under serum-free conditions, and measured the parameters of cardiac hypertrophy (see Fig. 1 for treatment protocol).
Upon inert adenovirus infection (Ad␤-gal ϩ AdtTA) the cells maintained their smaller size and moderate sarcomeric organization. By contrast, treatment with PE induced a significant increase in cell size and caused a reorganization of sarcomeres, as was shown by staining the sarcomers with anti-actinin antibody (Fig. 5A). Most importantly, adenoviral overexpression of MDM2 efficiently mitigated both cell size and sarcomeric organization in PE-stimulated cardiac myocytes (Fig.  5A). PE-induced sarcomer reorganization and increased cell size, however, was restored by blocking MDM2 expression upon the addition of tetracycline to the culture medium. Notably, we obtained similar results of reperfusion compared with the base-line values. *, significant difference from wild-type (wt) hearts (p Ͻ 0.05). D, infarct size is expressed as the percentage of the total myocardium detected with triphenyltetrazolium chloride staining. Data are averaged from five hearts. Significant difference from wild-type hearts: *, p Ͻ 0.05; **, p Ͻ 0.01). E, relative increase of lactate dehydrogenase leakage during reperfusion (compared with the corresponding base-line perfusion values). Samples of coronary effluents were collected during base-line perfusion and reperfusion. Data are averaged from five hearts. *, significant difference from wild-type samples (p Ͻ 0.01).

MDM2 Promotes Cardiomyocyte Survival but Blocks Hypertrophy
Next we determined the intensity of forced protein synthesis, which can be monitored by the uptake of a radiolabeled amino acid. Treatment with PE and ET-1 for 36 h increased the [ 3 H]leucine incorporation by 2.3-2.8-fold over the basal level (Fig. 5C). Similar to other parameters of hypertrophy, adenoviral overexpression of MDM2 almost completely prevented the elevated amino acid uptake associated with PE or ET-1 administration, and this effect was reversed upon blocking MDM2 expression by treating the cells with tetracycline (Fig. 5C).
In addition, the increased heart weight/body weight ratio in the hypomorphic compared with the wild-type mice was consistent with hypertrophy caused by lower MDM2 levels in adult animals (Fig. 5D). In fact, cardiac myocyte cross-sectional area from hypomorphic mice proved to be slightly but significantly increased when related to wild-type littermates (Fig. 5, E-F).
Together these results indicate that MDM2 inhibits the hypertrophic response of cardiac myocytes to PE and ET-1 stimulation, because MDM2 overexpression attenuated each investigated parameter of cardiomyocyte hypertrophy, including cell size, sarcomer formation, ANF expression, and amino acid uptake. Moreover, our in vivo measurements also support a role for MDM2 in the regulation of cardiac hypertrophy.

Do Terminally Differentiated Cardiac Myocytes Need MDM2
Activity?-MDM2 is a negative regulator of the tumor suppressor p53 and thereby represents a critical upstream factor to determine whether cells enter the cell cycle or die by apoptosis (9). In adult and neonatal cardiac myocytes, however, the activity of MDM2 might not be important either for cell cycle regulation or for cell survival because they are non-dividing, terminally differentiated cells (46) and undergo apoptosis in the absence of p53 in animal models of cardiac ischemia (40,41). The notion that MDM2 activity is dispensable for the adult/neonatal life of cardiac cells is negated by the current study, which demonstrates that the endogenous MDM2 constitutively regulates p53 activity and is required for maintaining the viability of both adult and neonatal cardiac myocytes. This was established by two different approaches involving the inhibition of MDM2 in isolated neonatal cardiac myocytes and cardiac ischemia/reperfusion studies on a mouse model with reduced MDM2 expression.
Role of MDM2 in Cardiomyocyte Survival during Ischemia/Reperfusion-In response to hypoxia, the protein level of p53 becomes elevated and mediates apoptosis in most cell systems (47). In cardiac myocytes, hypoxia also increases the level of p53 (48,49), but this is not necessarily required for apoptotic cell death. Although adenoviral delivery of p53 was sufficient to promote apoptosis of isolated cardiomyocytes (27), in chronic coronary artery ligation and the Langendorff ischemia/reperfusion models, endogenous p53 was dispensable for apoptotic cell death (40,41).
We, therefore, asked whether the p53 inhibitor MDM2 might protect cardiomyocytes against ischemic injury. Our results demonstrate that both endogenous and ectopically overexpressed MDM2 promote cell survival under several different experimental conditions. First, cultured cardiac myocytes proved to be less susceptible to hypoxia/reoxygenation when MDM2 was overexpressed. Second, treatment of isolated cardiomyocytes with an MDM2 inhibitor led to p53 stabilization and apoptosis. Finally, gradual loss of MDM2 expression in an animal model made the isolated mouse hearts more sensitive to ischemia/reperfusion injury with respect to functional performance as well as myocardial survival.
In addition, MDM2 should also inactivate p53-independent proapoptotic proteins, because it prevents apoptosis, unlike p53 deletion, which does not (50). In this regard, MDM2 might affect the transcriptional activity but not the stability of p73 (51). It may also stabilize p63 (51), inhibit retinoblastoma function on E2F1, and antagonize the apoptotic properties of E2F1 (52). Which of these pathways are actually players during cardiac ischemia/reperfusion in concert with p53 remains to be investigated.
Potential Cardiac Side Effects of Anticancer Therapy Based on p53 Reactivation-One of the most important challenges in designing tumor therapy is to achieve specific elimination of tumor cells while allowing the neighboring normal cells to survive. In particular, the application of several drugs as anticancer agents is limited by their toxic effects on the heart. For example, treatment with the DNA damaging agent doxorubicin leads to p53-induced cardiomyocyte apoptosis (30), and blockade of ErbB2 activity in cardiomyocytes by the inhibitory antibody herceptin activates the mitochondrial apoptotic pathway by changing the proportion of Bcl-x s and Bcl-x L (53).
Activation of the p53 pathway through inhibition of MDM2 by peptides and small molecules has recently been proposed as a novel therapeutic strategy for treating tumors carrying a wild-type p53 gene (54 -56). Although the development of such MDM2 inhibitors is an intensively investigated field, their potential cardiac toxicity has not yet been analyzed. In the present study, treatment with the MDM2 inhibitor PNC-28 led to p53 stabilization and apoptosis both during normoxia and hypoxia/reoxygenation. PNC-28 was previously published to be toxic to K-ras-transformed pancreatic cells at 100 g/ml without changing the viability of wild-type pancreatic and umbilical cord bloodderived stem cells (35). In our studies on cardiomyocytes, 100 g/ml PNC-28 caused significant cardiomyocyte death.
It, thus, appears that cardiac myocytes are sensitive to MDM2 inhibitor treatment, in particular during ischemia/reperfusion. Consequently, MDM2 inhibitors need to be tested for cardiac side effects before their introduction as anticancer drugs. Although MDM2 inhibitors allow the stabilization of p53 and activation of p53-dependent transcription and apoptosis, they might also cause p53-independent toxicity (35,57). Therefore, the contribution of p53-independent effects cannot be excluded in our experiments. Moreover, such a scenario were stained with anti-ANF (red) and anti-sarcomeric actinin (green) antibodies together with TO-PRO-3 (blue). Images were taken by confocal fluorescent microscope at 400ϫ magnification. B, percentage of cardiomyocytes expressing ANF. Myocytes were scored for the presence of perinuclear ANF staining in 10 randomly selected fields (at least 200 cells) from each group. C, amino acid uptake. 24 h before the end of the experiment, 1 Ci/ml [ 3 H]leucine was added to the cells, and incorporation was measured by a liquid scintillation counter. All data in A-C are averaged or are representatives from four experiments. The error bars are S.E. of the mean. *, significant difference from vehicle ϩ no virus samples (p Ͻ 0.01). #, significant difference from the corresponding MDM2 ϩ Tet samples (p Ͻ 0.01). D, heart weight/body weight ratio (mg/g) of hearts with different mdm2 genotypes. Values are calculated from data presented in Table 1. wt, wild type. E, hematoxylin and eosin staining of histologic sections from wild-type and mdm2 puro/del7-9 mouse hearts. Hearts were processed as detailed in "Experimental procedures." F, cardiac myocyte cross-sectional area in hearts (n ϭ 3) with different mdm2 genotypes. Cross-sectional area was determined by measuring at least 200 cells per heart stained with hematoxylin and eosin. *, p Ͻ 0.05, significant difference from wild-type hearts.
would be consistent with the view that, in addition to p53, MDM2 regulates the level and activity of other proteins involved in cell survival (9,23).
Role of MDM2 in Cardiac Hypertrophy-A variety of different protein kinase cascades have been implicated in the regulation of cardiac hypertrophy. Among these, the Akt pathway plays an essential role both in normal and hypertrophic growth. Akt exerts its effect primarily through mammalian target of rapamycin and glycogen synthase kinase-3, initiating a hypertrophic program that includes activation of protein synthesis and reprogramming of gene expression by a new set of transcription factors (44,45,58). Because MDM2 is stabilized by Aktdriven phosphorylation (13)(14)(15), we expected that MDM2 either does not affect hypertrophy or is one of the prohypertrophic targets of Akt. In contrast, our results indicate that the survival protein MDM2 attenuates cardiomyocyte hypertrophy induced by the ␣-agonists phenylephrine or endothelin-1. Importantly, hypomorphic MDM2 mice demonstrated smaller hearts (likely due to higher rate of apoptosis) but bigger individual cardiac myocytes (likely due to enhanced hypertrophy) than their wild-type littermates. This is a unique effect, because most survival proteins also promote cardiac hypertrophy (44, 45, 59 -61). Because MDM2 is an E3 ubiquitin ligase, it is reasonable to assume that MDM2 promotes proteasomal degradation of a prohypertrophic component of the hypertrophy regulatory network, and down-regulation of this currently unidentified protein leads to decreased hypertrophy. Because Akt promotes hypertrophy and MDM2 is antihypertrophic, MDM2 may provide a negative feedback control to modify the Akt effect. The target of MDM2 in the hypertrophy pathway, however, does not seem to be p53, because MDM2 suppresses both p53 levels and hypertrophy. The identity of this MDM2 target protein is currently under investigation in our laboratory.
In summary, our studies describe a unique combination of prosurvival and antihypertrophic effects of MDM2 in cardiomyocytes. Activation of MDM2 may, thus, provide a dual beneficial therapeutic effect by promoting cardiomyocyte survival without leading to decompensated hypertrophy in the infarcted heart. Therefore, MDM2 may serve as a novel cardiac gene therapy target. In addition, our data also suggest that anticancer drugs acting through MDM2 inhibition might provoke adverse cardiac side effects.