Published online before print December 22, 2008, doi: 10.1161/CIRCULATIONAHA.108.799700
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(Circulation. 2009;119:99-106.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
Acute Doxorubicin Cardiotoxicity Is Associated With p53-Induced Inhibition of the Mammalian Target of Rapamycin Pathway
From the Riley Heart Research Center, Herman B. Wells Center for Pediatric Research (W.Z., M.H.S., H.C., W.S., R.M.P., E.A.L., R.L.C., W.S., L.J.F.), and the Krannert Institute of Cardiology (L.J.F.), Indiana University School of Medicine, Indianapolis, Ind.
Correspondence to Loren Field, Wells Center, 1044 W Walnut St, R4 Bldg, Room W376, Indianapolis, IN 46202-5225. E-mail ljfield{at}iupui.edu
Received June 16, 2008; accepted October 17, 2008.
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Abstract |
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Background— Doxorubicin is used to treat childhood and adult cancer. Doxorubicin treatment is associated with both acute and chronic cardiotoxicity. The cardiotoxic effects of doxorubicin are cumulative, which limits its chemotherapeutic dose. Free radical generation and p53-dependent apoptosis are thought to contribute to doxorubicin-induced cardiotoxicity.
Methods and Results— Adult transgenic (MHC-CB7) mice expressing cardiomyocyte-restricted dominant-interfering p53 and their nontransgenic littermates were treated with doxorubicin (20 mg/kg cumulative dose). Nontransgenic mice exhibited reduced left ventricular systolic function (predoxorubicin fractional shortening [FS] 61±2%, postdoxorubicin FS 45±2%, mean±SEM, P<0.008), reduced cardiac mass, and high levels of cardiomyocyte apoptosis 7 days after the initiation of doxorubicin treatment. In contrast, doxorubicin-treated MHC-CB7 mice exhibited normal left ventricular systolic function (predoxorubicin FS 63±2%, postdoxorubicin FS 60±2%, P>0.008), normal cardiac mass, and low levels of cardiomyocyte apoptosis. Western blot analyses indicated that mTOR (mammalian target of rapamycin) signaling was inhibited in doxorubicin-treated nontransgenic mice but not in doxorubicin-treated MHC-CB7 mice. Accordingly, transgenic mice with cardiomyocyte-restricted, constitutively active mTOR expression (MHC-mTORca) were studied. Left ventricular systolic function (predoxorubicin FS 64±2%, postdoxorubicin FS 60±3%, P>0.008) and cardiac mass were normal in doxorubicin-treated MHC-mTORca mice, despite levels of cardiomyocyte apoptosis similar to those seen in doxorubicin-treated nontransgenic mice.
Conclusions— These data suggest that doxorubicin treatment induces acute cardiac dysfunction and reduces cardiac mass via p53-dependent inhibition of mTOR signaling and that loss of myocardial mass, and not cardiomyocyte apoptosis, is the major contributor to acute doxorubicin cardiotoxicity.
Key Words: heart failure • apoptosis • myocytes
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Introduction |
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Anthracyclines such as doxorubicin, daunomycin, epirubicin, and idarubicin are widely used and highly successful anticancer chemotherapeutic drugs. Unfortunately, these drugs also induce acute cardiotoxicity, which is characterized by hypotension, tachycardia, arrhythmia, and transient depression of left ventricular function.1–4 In addition, high cumulative doses are associated with late-onset cardiomyopathy that is refractory to standard treatment. It is widely thought that free radical–induced mitochondrial damage contributes to doxorubicin-induced cardiotoxicity.5 In addition, doxorubicin can induce DNA damage, inhibit DNA and protein synthesis, promote myofiber degeneration, inhibit transcription of specific gene programs, and induce cardiomyocyte apoptosis via a caspase-3–dependent mechanism. Because doxorubicin can interfere with many different intracellular processes, it has proven difficult to determine the molecular mechanism of its acute and chronic cardiotoxicity.
Clinical Perspective p 106
Numerous studies have shown that doxorubicin-induced cardiomyocyte apoptosis is associated with increased expression of the p53 tumor suppressor protein. Moreover, reduction of p53 activity via genetic deletion6 or chemical inhibition7 is cardioprotective during short-term doxorubicin treatment. To further characterize the role of p53 in acute doxorubicin-induced cardiotoxicity, MHC-CB7 mice (which express dominant-interfering p53 in cardiomyocytes)8 were studied 7 days after the initiation of treatment. Cardiac function was improved, with a concomitant reduction in cardiomyocyte apoptosis, in the MHC-CB7 mice compared with their doxorubicin-treated nontransgenic siblings. Surprisingly, expression of the MHC-CB7 transgene also markedly blunted the doxorubicin-induced reduction of cardiac mass observed in nontransgenic mice. Western blot analyses indicated that doxorubicin treatment reduced the level of activated mammalian target of rapamycin (mTOR) in nontransgenic mice. mTOR is a serine/threonine protein kinase that regulates protein translation and cell growth.9 Expression of the MHC-CB7 transgene blocked doxorubicin-induced reduction of mTOR activity. To establish the role of mTOR signaling in doxorubicin-induced cardiotoxicity, mice expressing constitutively active mTOR in the myocardium (MHC-mTORca mice)10 were subjected to doxorubicin treatment. Expression of the MHC-mTORca transgene was sufficient to block doxorubicin-induced cardiac dysfunction and mass reduction but not cardiomyocyte apoptosis. These data suggest that acute doxorubicin-induced cardiotoxicity results from p53-dependent dysregulation of the mTOR pathway and that cardiomyocyte apoptosis does not contribute significantly to cardiac dysfunction during the immediate response to doxorubicin treatment.
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Methods |
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Mice
The present study used MHC-CB7 mice8 (n=56), MHC-mTORca mice10 (n=50), and their nontransgenic littermates (n=136). Mice were maintained in a DBA/2J genetic background. Adult mice received doxorubicin (2 intraperitoneal injections of 10 mg/kg at 3-day intervals, 20 mg/kg cumulative dose) or vehicle (saline) and were euthanized 7 days after the initial injection. All animal protocols were approved by the Indiana University School of Medicine Institutional Animal Care and Research Advisory Committee.
Echocardiography
Mice were lightly anesthetized with 1.5% isoflurane until the heart rate stabilized at 400 to 500 beats per minute. Two-dimensional short-axis images were obtained with a high resolution Micro-Ultrasound system (Vevo 770, VisualSonics Inc, Toronto, Canada) equipped with a 40-MHz mechanical scan probe. Fractional shortening (FS), ejection fraction, left ventricular internal diameter (LVID) during systole, LVID during diastole, end-systolic volume, and end-diastolic volume were calculated with Vevo Analysis software (version 2.2.3) as described previously.10 LVID during systole and during diastole was measured from M-mode recording at the level of the mid-papillary muscle, whereas end-systolic and end-diastolic volumes were measured with B-mode recording in a plane containing the aortic and mitral valves.
Histology
Hearts were harvested, cryoprotected in 30% sucrose, embedded, and sectioned at 10 µm by use of standard techniques. To quantify minimal cardiomyocyte fiber diameter, images from Sirius red/fast green–stained sections were captured, digitized, and analyzed with NIH Image 1.36b software as described previously.11 At least 400 randomly selected cardiomyocytes from each animal were analyzed. To quantify cardiomyocyte apoptosis, 4 transverse sections from each heart, sampled from the midpoint between the apex and base, were postfixed in 4% paraformaldehyde and screened for anti-activated caspase-3 immune reactivity (antibody #G7481, Promega, Madison, Wis), followed by a horseradish peroxidase–conjugated secondary antibody; signal was visualized with a diaminobenzidine reaction as described previously.8
Western Blot Analyses
Proteins were extracted and quantified by the Coomassie Blue method (Pierce, Rockford, Ill) as described previously.8 Samples were solubilized in SDS-PAGE loading buffer for 5 minutes at 95°C and resolved on 7% or 10% SDS-PAGE gels. Fractionated proteins were then electrotransferred from the gel to nitrocellulose (Amersham, Chalfont St-Giles, United Kingdom) filters in Towbin buffer at 200-mA constant current and analyzed by Western blotting. The filters were stained with 0.1% naphthol blue-black in 45% methanol, 10% acetic acid to assess the efficiency of transfer. Antibodies used recognized PARP (poly ADP-ribose polymerase; catalogue #9542, Cell Signaling Technology, Danvers, Mass), Bcl-xL (catalogue #sc-7195, Santa Cruz Biotechnology, Santa Cruz, Calif), p53 (catalogue #PC-35, EMD Chemicals, Gibbstown, NJ), AU1 tag (catalogue #MMS-130R, Covance Inc, Emeryville, Calif), P-mTOR[ser2481] (catalogue #2974, Cell Signaling Technology), P-mTOR[ser2448] (catalogue #2971, Cell Signaling Technology), total mTOR (catalogue #2972, Cell Signaling Technology), P-eIF4G[ser1108] (catalogue #2441, Cell Signaling Technology), total eIF4G (catalogue #SC-11373, Santa Cruz Biotechnology), P-eIF4E[ser209] (catalogue #9741, Cell Signaling Technology), total eIF4E (catalogue #9742, Cell Signaling Technology), 4EBP1 (catalogue #9452, Cell Signaling Technology), P-P70S6 kinase[thr421/ser424] (catalogue #9204, Cell Signaling Technology), total P70S6 kinase (catalogue #9202, Cell Signaling Technology), and total TSC2 (catalogue #SC-893, Santa Cruz Biotechnology). Signal was visualized by the enhanced chemiluminescence (ECL) method according to the manufacturer’s protocol (Amersham). Western signal was digitized and quantified with ImageJet software.
Statistical Analysis
All values are presented as mean±SEM. Statistical significance (P<0.01) was determined by Student t test (for groups of 2) or by 1-way ANOVA with Bonferroni correction (P<0.008 was considered significant; 6 pairwise comparisons).
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Doxorubicin Induces Acute Cardiotoxicity via a p53-Dependent Pathway
MHC-CB7 mice (which express dominant-interfering p53) and their nontransgenic littermates were used to study the role of p53 in acute doxorubicin cardiotoxicity. Before doxorubicin administration, left ventricular systolic function was similar in nontransgenic and MHC-CB7 mice (Figure 1A; online-only Data Supplement Table I). Seven days after the initiation of doxorubicin treatment, FS was markedly reduced in the nontransgenic mice (Figure 1A). Increased LVID during systole and increased end-systolic volume were also noted (online-only Data Supplement Table I). In contrast, left ventricular systolic function and cardiac dimensions in MHC-CB7 mice were not altered by doxorubicin treatment (Figure 1A; online-only Data Supplement Table I). Representative echocardiograms from the nontransgenic and MHC-CB7 mice are shown in Figure 1B.
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Activated caspase-3 was used as a marker to quantify cardiomyocyte apoptosis. Because activated caspase-3 immune reactivity is present in the cytoplasm, cardiomyocytes at early stages of apoptosis can be identified in histological sections based on cell size and shape.8 Doxorubicin treatment resulted in a marked increase in the number of activated caspase-3 immune-reactive cardiomyocytes in nontransgenic animals (Figure 2A). Although doxorubicin treatment induced cardiomyocyte activated caspase-3 immune reactivity in MHC-CB7 mice, the level of induction was approximately 4-fold reduced compared with doxorubicin-treated nontransgenic mice (Figure 2A). Cleavage of PARP, a substrate of caspase-3, was also monitored. Doxorubicin treatment in nontransgenic mice resulted in PARP cleavage (Figure 2B, upper panel), consistent with the observed increase in activated caspase-3 immune reactivity. In contrast, PARP cleavage product was not detected in doxorubicin-treated MHC-CB7 mice. Expression of the MHC-CB7 transgene was associated with a modest induction of Bcl-xL, a prosurvival member of the Bcl-2 gene family (Figure 2B). Expression of several other Bcl-2 family members (Bax, Bak, and Bcl-2) was not altered (data not shown). Anti-p53 Western blots confirmed expression of the MHC-CB7 transgene (Figure 2B). The antibody used recognizes both CB7 and wild-type p53; in agreement with other studies,7 increased levels of endogenous p53 were observed in longer exposures of Western blots from doxorubicin-treated nontransgenic hearts (Figure 2B, lower panel).
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P<0.008 vs doxorubicin-treated nontransgenic mice; n=5 mice per group. B, Western blot analysis of apoptosis-related proteins in nontransgenic and MHC-CB7 mice after saline or doxorubicin treatment (see the online-only Data Supplement for densitometric signal quantitation; short and long exposures of the anti-p53 blot are shown). Exp indicates exposure.
Doxorubicin treatment had a dramatic impact on heart size in nontransgenic mice. Total heart weight was reduced by 
30% (Figure 3A; a reduction in body weight was also noted, as shown in Table II in the online-only Data Supplement). Previous studies have shown that cardiomyocyte minimal fiber diameter measurements can be used to quantify myocyte hypertrophy and atrophy.12 A reduction in cardiomyocyte minimal fiber diameter was apparent after doxorubicin treatment of nontransgenic mice, which suggests that the reduction in heart weight largely reflected doxorubicin-induced reduction of cardiomyocyte size (Figure 3B; online-only Data Supplement Table II). In contrast, heart weight and cardiomyocyte minimal fiber diameter in MHC-CB7 mice were largely unaffected by doxorubicin treatment (Figure 3A and 3B), although a marked reduction in body weight was observed (online-only Data Supplement Table II). Control dose-response experiments with nontransgenic mice revealed that the onset of decreased cardiac mass and cardiomyocyte apoptosis (as indicated by PARP cleavage) were first detected at the same dose of doxorubicin (Figure VI, online-only Data Supplement). Collectively, these data indicate that acute doxorubicin-induced cardiac dysfunction, cardiomyocyte apoptosis, and cardiac mass reduction occur via a p53-dependent pathway, and these characteristics of acute doxorubicin-induced cardiotoxicity can effectively be blocked by expression of dominant-interfering p53.
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Doxorubicin Alters mTOR Pathway Activity via a p53-Dependent Mechanism
The marked heart weight reduction in doxorubicin-treated nontransgenic mice was somewhat surprising. Previous studies have shown that the mTOR pathway plays a critical role in the regulation of protein translation and cell growth.9 We therefore compared the activity and total level of mTOR in hearts from saline- and doxorubicin-treated nontransgenic mice. mTOR undergoes an autophosphorylation event at serine residue 2481, and the presence of P-mTOR[ser2481] is often used as an indicator of mTOR activity.13 Doxorubicin treatment resulted in an approximately 4-fold reduction of P-mTOR[ser2481] levels in the hearts of nontransgenic mice but did not impact total mTOR levels (Figure 4; see the online-only Data Supplement for densitometric quantification). In contrast, no changes in P-mTOR[ser2481] levels were observed in doxorubicin-treated MHC-CB7 hearts (Figure 4).
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To determine the potential consequences of decreased levels of active mTOR, the canonical downstream effectors were analyzed. No changes in the total level or phosphorylation status of 4EBP1 and p70S6 kinase were apparent in doxorubicin-treated nontransgenic hearts (Figure VII, online-only Data Supplement). mTOR activation also promotes the phosphorylation of protein translation initiation factor eIF4G at serine residue 1108 (P-eIF4G[ser1108]), as well as the dephosphorylation of eIF4E at serine residue 209 (P-eIF4E[ser209]). These posttranslational modifications have been shown to enhance protein translation initiation under some experimental circumstances (see Discussion). Doxorubicin treatment decreased P-eIF4G[ser1108] levels and increased P-eIF4E[ser209] levels in nontransgenic mice, without impacting the total level of the initiation factors (Figure 4). In contrast, no changes were apparent in P-eIF4G[ser1108] or P-eIF4E[ser209] levels in doxorubicin-treated MHC-CB7 hearts (Figure 4).
Constitutively Active mTOR Blocks Acute Doxorubicin Cardiotoxicity
The data presented above suggest that acute doxorubicin cardiotoxicity occurs via p53-dependent modulation of mTOR signaling. To directly test this hypothesis, we used transgenic mice that express a mutant mTOR in which the autoinhibition domain located between amino acid residues 2430 and 2450 was deleted.14 These animals (designated MHC-mTORca) exhibit constitutively elevated mTOR activity in the heart.10 An AU1 epitope tag was engineered in the N-terminus to facilitate identification of the transgene-encoded protein via Western blot analysis. Adult MHC-mTORca mice and their nontransgenic littermates were injected with doxorubicin and subjected to functional, histological, and molecular analyses as described above. Doxorubicin treatment resulted in a marked reduction in FS in nontransgenic mice but had no impact on cardiac function in the MHC-mTORca animals (Figure 5). Increased LVID during systole was apparent in the doxorubicin-treated nontransgenic mice but not in the doxorubicin-treated MHC-mTORca mice (Table III, online-only Data Supplement).
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Cardiomyocyte apoptosis was quantified by activated caspase-3 immune reactivity. As observed above, doxorubicin treatment resulted in a marked increase in the level of cardiomyocyte activated caspase-3 immune reactivity in nontransgenic mice (Figure 6A). Surprisingly, comparable levels were observed in doxorubicin-treated nontransgenic and doxorubicin-treated MHC-mTORca animals. Western blot analyses revealed similar levels of cleaved PARP in the doxorubicin-treated nontransgenic and MHC-mTORca mice (Figure 6B, upper panel), consistent with the activated caspase-3 results. Moreover, no induction of Bcl-xL was observed in the transgenic hearts. Transgene expression was confirmed via Western analysis with an anti-AU1 antibody. A 26% reduction in heart weight and a 12% reduction in cardiomyocyte minimal fiber diameter were observed in doxorubicin-treated nontransgenic mice (Figure 7A and 7B; Table IV, online-only Data Supplement). In contrast, doxorubicin treatment had no impact on these parameters in MHC-mTORca mice. Tritiated thymidine incorporation analyses11 indicated that doxorubicin treatment did not induce cell cycle activity in the MHC-mTORca mice (data not shown); thus, cardiomyocyte replacement cannot account for the preservation of cardiac mass. Interestingly, normal cardiac function and mass were retained for as long as 3 weeks after doxorubicin treatment in the MHC-mTOR mice (Table V, online-only Data Supplement). Collectively, these data indicate that activation of the mTOR pathway effectively blocks acute doxorubicin-induced cardiac dysfunction and mass reduction but not acute doxorubicin-induced cardiomyocyte apoptosis.
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Western blot analyses were performed to determine the impact of MHC-mTORca transgene expression on posttranslational modification of the downstream effectors of the mTOR signaling pathway. As observed above, doxorubicin treatment in nontransgenic mice resulted in decreased levels of P-mTOR[ser2481] and P-eIF4G[ser1108] and increased levels of P-eIF4E[ser209] (Figure 8; see the online-only Data Supplement for densitometric quantification). Expression of the MHC-mTORca transgene resulted in an increase in total mTOR levels, as well as an approximately 2-fold increase in the level of P-mTOR[ser2481]. This constitutive mTOR activity resulted in increased levels of P-eIF4G[ser1108] and decreased levels of P-eIF4E[ser209] in saline-treated animals. Doxorubicin treatment had no impact on the levels of these phosphorylation events in MHC-mTORca mice (Figure 8).
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Discussion |
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The data presented here support a model wherein acute doxorubicin-induced cardiac systolic dysfunction and cardiac mass loss occur via p53-dependent modulation of the mTOR pathway. The data also demonstrate that acute doxorubicin-induced cardiomyocyte apoptosis occurs via a p53 dependent pathway but is independent of mTOR signaling. The presence of similar levels of cardiomyocyte activated caspase-3 immune reactivity in doxorubicin-treated nontransgenic and MHC-mTORca hearts suggests that cardiomyocyte apoptosis per se does not contribute significantly to decreased cardiac function during acute doxorubicin cardiotoxicity. Rather, inhibition of the mTOR signaling pathway appears to be the predominant contributor to acute doxorubicin cardiotoxicity.
Previous studies have implicated the p53 pathway in doxorubicin-induced cardiotoxicity. For example, reduced levels of cardiomyocyte apoptosis and concomitant improvements in cardiac function were observed in doxorubicin-treated p53 null mice compared with their wild-type littermates.6 Similarly, pretreatment with pifithrin-
(a chemical that inhibits nuclear translocation of p53) reduced doxorubicin-induced cardiomyocyte apoptosis and cardiac dysfunction in vivo.7 Thus, the reduced level of cardiomyocyte apoptosis observed in doxorubicin-treated MHC-CB7 mice was not unexpected. This phenotype may result in part from the concomitant, albeit modest, increase in steady state levels of Bcl-xL (Figure 2), because other studies have demonstrated that p53 can regulate cell apoptosis in part by interacting with Bcl-xL.15
In contrast, the impact of p53 inactivation on doxorubicin-induced cardiac mass reduction was not expected, nor was it noted in previous studies. The observation that doxorubicin treatment decreased mTOR activity (as evidenced by decreased P-mTOR[ser2481] levels) in nontransgenic mice but not in MHC-CB7 mice, coupled with the marked cardioprotection observed in doxorubicin-treated MHC-mTORca mice, strongly implicates a causal relationship between p53 and mTOR signaling during acute doxorubicin-induced cardiotoxicity. Indeed, recent studies in noncardiomyocytes have shown that p53 can negatively regulate the mTOR pathway via the tuberous sclerosis complex (TSC) 2-RHEB G-protein axis.16–19 Although doxorubicin treatment failed to alter steady state levels of TSC2 in hearts from doxorubicin-treated mice (Figure VII, online-only Data Supplement), a recent study suggests that posttranslational modifications might be more relevant for regulation of TSC2 activity in muscle.20
The mTOR pathway has been implicated previously in the regulation of cardiomyocyte growth. For example, alcohol decreases the level of mTOR phosphorylation and activity in the myocardium, with a concomitant reduction of cardiac mass. Interestingly, phosphorylation was reduced predominantly at P-mTOR[ser2481] after short-term alcohol administration21 and predominantly at P-mTOR[ser2448] after sustained alcohol administration.22 mTOR signaling is also important for the induction of cardiac hypertrophy. Physiological cardiac hypertrophy induced by treadmill exercise is associated with activation of the mTOR pathway.23 Pathophysiological cardiac hypertrophy induced by aortic banding is also associated with rapamycin-sensitive activation of mTOR at early, but not late, time points.23,24 Thyroid hormone–induced hypertrophy also activates mTOR signaling and is rapamycin sensitive.25
Although these studies indicate that mTOR is important for the regulation of myocardial growth, they also illustrate that its signaling through downstream effector molecules is very complex and at times contradictory. This fact is underscored by the absence of overt changes in the canonical mTOR downstream effectors p70S6 kinase and 4EBP-1 in hearts from doxorubicin-treated nontransgenic mice (Figure VII, online-only Data Supplement). In this regard, it is of interest to note that previous studies using nonphosphorylatable point mutants have shown that dephosphorylation at mTOR serine residue 2448 (but not 2481) mediates rapamycin-induced inhibition of p70S6 kinase and 4EBP-1 activity.13,26 Indeed, marked dephosphorylation of mTOR serine residue 2448 and concomitant reduction of p70S6 kinase and 4EBP-1 phosphorylation were observed in hearts from rapamycin-treated mice but not in those from doxorubicin-treated mice (Figure VII, online-only Data Supplement). Thus, the present data support the previous studies indicating that posttranslational modulation of mTOR residue 2448 is responsible for regulation of p70S6 kinase and 4EBP-1 activity. Conversely, the observation that coadministration of doxorubicin and rapamycin did not result in reduced cardiac mass in MHC-CB7 mice and did not further exacerbate doxorubicin-induced cardiac mass in nontransgenic mice (online-only Data Supplement Table VI) supports the importance of mTOR residue 2481 in acute doxorubicin-induced cardiotoxicity. The complexity of feedback regulation of the mTOR pathway is further underscored by the absence of overt cardiac hypertrophy in the MHC-mTORca mice (Figure 7) and the absence of a reduction in baseline cardiac mass in rapamycin-treated rats.25
The observed changes in P-eIF4G[ser1108] and P-eIF4E[ser209] levels in doxorubicin-treated nontransgenic mice, coupled with the absence of changes in doxorubicin-treated MHC-CB7 and MHC-mTORca mice, raises the possibility that these molecules may be downstream mTOR effectors during acute doxorubicin cardiotoxicity. eIF4G is a scaffold protein that links eIF3 and the 43S initiation complex.27,28 Serum stimulation promotes rapamycin-sensitive increases in P-eIF4G[ser1108] and concomitant protein synthesis in cultured HEK293 cells.29 Moreover, rats exhibit rapamycin-sensitive increases in P-eIF4G[ser1108] in the myocardium after controlled food intake.30 Thus, mTOR-mediated phosphorylation (either directly or indirectly) of eIF4G at serine residue 1108 is associated with anabolic growth in the heart. Of interest, activation of a temperature-sensitive p53 transgene in murine erythroleukemia cells decreased P-mTOR[ser2481] and P-eIF4G[ser1108] levels and promoted cellular atrophy,31 a sequela that is remarkably similar to that observed in doxorubicin-treated myocardium.
The situation with eIF4E is less straightforward. eIF4E binds to the 5' end cap structure of the mRNA and interacts with eIF4G.27,28 Phosphorylation of eIF4E at serine residue 209 is rapamycin sensitive32 and has been reported to inhibit,33 enhance,34 or have no impact35 on protein translation, depending on the cell type and experimental system used. P-eIF4E[ser209] has reduced affinity for capped mRNA, which is thought to promote the release of initiation factors from existing translational complexes.36 Such activity could contribute in part to the reduction in cardiac mass observed in doxorubicin-treated hearts. Ultimately, direct proof of the role of these (or any other) putative mTOR effector molecules in doxorubicin-induced cardiotoxicity will require the generation of additional transgenic models with the relevant phosphomimetic and/or nonphosphorylatable mutations. However, as with all transgenic overexpression studies, the possibility that elevated mTOR activity is cardioprotective irrespective of the molecular cause of doxorubicin-induced ventricular induction in nontransgenic animals constitutes a limitation of the present study.
The p53/mTOR axis may regulate other pathways that contribute to acute doxorubicin cardiotoxicity. For example, activation of the ubiquitin-proteasome system has been reported in doxorubicin-treated cardiomyocytes.37 Interestingly, proteasome-mediated degradation of the p300 transcription coactivator has been implicated in negative regulation of myocardial transcription programs.38–40 Activation of the ubiquitin-proteasome system has also been implicated in heart size reduction after chronic unloading,41,42 albeit with a paradoxical increase in mTOR activity. It is also tempting to speculate that cardiomyocyte autophagy43,44 may contribute to acute doxorubicin cardiotoxicity. Although preliminary analysis failed to establish direct correlations between the presence of molecular markers for proteasome and/or autophagy activity and the presence of cardiotoxicity in doxorubicin-treated mice under the conditions used here (W.Z., unpublished observations, 2008), the potential role of these pathways in acute doxorubicin cardiotoxicity cannot be discounted.
In summary, the data reported here suggest that acute doxorubicin-induced cardiotoxicity results from p53-dependent modulation of mTOR activity. The data also suggest that cardiomyocyte apoptosis does not contribute to decreased cardiac function at the time point studied. It has recently been reported that treatment with erythropoietin,45 thrombopoietin,46 or granulocyte colony-stimulating factor47 can blunt doxorubicin-induced cardiac dysfunction and mass reduction. It would be informative to determine whether these molecules also work via alteration of mTOR signaling. Additionally, it would be of considerable interest to determine whether upstream effectors that activate the mTOR pathway would be cardioprotective in the setting of acute doxorubicin cardiotoxicity.
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Acknowledgments |
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Sources of Funding
This work was supported by grants from the National Heart, Lung, and Blood Institute.
Disclosures
None.
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CLINICAL PERSPECTIVE
Anthracyclines are widely used and highly successful anticancer chemotherapeutic drugs. Unfortunately, these drugs also cause acute cardiotoxicity, such as arrhythmia and transient depression of left ventricular function, and higher cumulative doses are associated with late-onset refractory cardiomyopathy and heart failure. Because anthracyclines can interfere with many different intracellular processes, it has proven difficult to determine the exact mechanisms that give rise to acute versus chronic cardiotoxicity. This study examined acute cardiotoxicity in mice treated with the anthracycline doxorubicin. Treated mice exhibited a marked decrease in cardiac function acutely that was accompanied by a reduction in cardiac mass. Molecular analyses suggested that doxorubicin treatment increased levels of the tumor suppressor protein p53, which in turn inhibited the activity of mammalian target of rapamycin (mTOR). Inhibition of mTOR activity is predicted to decrease the translation of new proteins. In support of this mechanism, genetically modified mice expressing either dominant-interfering p53 or constitutively active mTOR exhibited normal cardiac function and mass after doxorubicin treatment. Interestingly, rescue of cardiac function and structure during acute cardiotoxicity was not associated with reduction of doxorubicin-induced cardiomyocyte apoptosis. These data suggest that coadministration of mTOR activators might decrease acute doxorubicin cardiotoxicity, thereby permitting a higher dose of drug per treatment. If so, administration of fewer doses of anthracycline at higher levels might be a more effective anticancer strategy. This approach may also allow lower cumulative doses, which would reduce the incidence of late-onset refractory cardiomyopathy.
Circulation 2009 119: 1-4.
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