Cardiotoxicity of the cancer therapeutic agent imatinib mesylate

Risto Kerkelä^1, 2, Luanda Grazette³, Rinat Yacobi⁴, Cezar Iliescu⁵, Richard Patten², Cara Beahm¹, Brian Walters², Sergei Shevtsov^1, 2, Stéphanie Pesant¹, Fred J Clubb⁶, Anthony Rosenzweig³, Robert N Salomon⁷, Richard A Van Etten⁴, Joseph Alroy^7, 8, Jean-Bernard Durand⁵ & Thomas Force^1, 2

¹ Center for Translational Medicine, Jefferson Medical College, 1025 Walnut Street, Philadelphia, Pennsylvania 19107, USA.

² The Molecular Cardiology Research Institute, Tufts–New England Medical Center and Tufts University School of Medicine, 750 Washington Street, Boston, Massachusetts 02111, USA.

³ Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, USA.

⁴ Molecular Oncology Research Institute, Tufts–New England Medical Center and Tufts University School of Medicine, 750 Washington Street, Boston, Massachusetts 02111, USA.

⁵ M.D. Anderson Cancer Center and The University of Texas, 1515 Holcombe Boulevard Houston, Texas 77030, USA.

⁶ Department of Cardiovascular Pathology, Texas Heart Institute, 6770 Bertner Avenue Houston, Texas 77030, USA.

⁷ Department of Pathology, Tufts–New England Medical Center and Tufts University School of Medicine, 750 Washington Street, Boston, Massachusetts 02111, USA.

⁸ Tufts Cummings School of Veterinary Medicine, 200 Westboro Road, North Grafton, Massachusetts 01536, USA.

Here we report ten individuals who presented with severe CHF without obvious cause while receiving imatinib. We found that imatinib has deleterious effects on cardiomyocytes in culture and in vivo. The triggering mechanism seems to be activation of the ER stress response (also known as the unfolded protein response), a response that protects cells by shutting down general protein translation (by PKR-like ER kinase (PERK)-mediated phosphorylation of the translation initiation factor eIF2) while upregulating the expression of specific protective stress response genes. If ER stress is prolonged, however, prodeath pathways including Jun N-terminal kinases (JNKs) are activated. We show that these pathways are activated in cardiomyocytes exposed to imatinib, leading to profound alterations of mitochondrial function and to cardiomyocyte death.

We collected clinical data from ten individuals who developed significant left ventricular dysfunction during their course of therapy with imatinib (Supplementary Table 1 online). All ten individuals had normal left ventricular function before imatinib therapy was instituted (ejection fraction 56 7%), but presented after a mean of 7.2 5.4 months (range 1–14 months) of therapy with heart failure, including significant volume overload and symptoms corresponding to a New York Heart Association (NYHA) class 3–4 heart failure (mean NYHA class 3.5 0.5; P < 0.001 versus pretreatment). Ejection fraction by radionuclide imaging at presentation was 25 8% (P < 0.001 versus pretreatment ejection fraction; Fig. 1a). This low ejection fraction was associated with mild left ventricular dilation (mean left ventricular end-diastolic diameter 57.8 4.8 mm at presentation; range, 52–65 mm; normal range 35–56 mm; ref. 16).

Figure 1. Imatinib is cardiotoxic in humans. (a) Change in left ventricular ejection fraction (LVEF) from pretreatment to presentation with heart failure while on imatinib. The ejection fraction was determined by radionuclide imaging before initiation of imatinib therapy and again at presentation with CHF. *P < 0.001 versus pretreatment value. (b) Electron micrographs of cardiac biopsies. Shown are electron micrographs from individuals who presented with presumed imatinib-induced heart failure. Left, arrow indicates a dense membrane whorl. Right, arrow indicates cardiomyocyte mitochondria with partially effaced cristae. Arrowheads indicate dilated sarcoplasmic reticulum with membrane whorls. Asterisk indicates a cardiomyocyte with effaced myofilaments and glycogen accumulation. Scale bars, 2 m. Full Figure and legend (28K)

Figure 2. Imatinib is cardiotoxic in mice. Ultrastructural analysis and assay for mitochondrial permeability transition pore opening. (a) Electron micrographs of hearts from mice treated with intraperitoneal injections of vehicle (left) or imatinib (50 mg/kg/d; right) for 3 weeks. Top panels, sarcoplasmic reticulum and vacuoles containing membrane whorls (arrows). Middle right, a vacuole with a membrane whorl within or immediately adjacent to mitochondria. Bottom right, pleomorphic mitochondria in the imatinib-treated hearts. None of these findings was present in the vehicle-treated hearts. (b) Imatinib enhances Ca2+-induced mitochondrial swelling. Mitochondria were isolated from hearts of mice treated with either imatinib (200 mg/kg/d) or vehicle for 5 weeks. Swelling, as determined by light scattering, was measured after the addition of 250 M Ca2+. Studies were also done in the presence of cyclosporin A (CsA; 10 mg/ml), an inhibitor of cyclophilin D that blocks opening of the permeability transition pore. (c) Hematoxylin and eosin stain of a heart from a mouse treated with either vehicle (left) or imatinib (200 mg/kg/d orally; right) for 5 weeks. Original magnification, 40. Full Figure and legend (66K)

Table 1. Echocardiographic indices of imatinib-treated mice Full Table

Figure 3. Effect of imatinib treatment on mitochondrial function and on mediators and markers of apoptosis. (a) Imatinib induces loss of mitochondrial membrane potential (m). NRVMs were treated with vehicle (left) or imatinib (5 M, right; doses indicated below) for 18 h, and were then loaded with either MitoTracker Red (top) or JC-1 (bottom). MitoTracker Red accumulates in mitochondria in which m is maintained and is released into the cytosol when m collapses. Punctate (mitochondrial) staining was lacking in the imatinib-treated cells. For JC-1, a decrease in the red/green ratio is a measure of loss of m. *P < 0.01 versus control, **P < 0.001 versus control. Original magnification, 40. (b) Imatinib induces cytochrome c release. NRVMs were treated with imatinib (5 M, right) or vehicle (control; left) for 24 h and were fixed and stained for cytochrome c (top panels). There was marked release of cytochrome c as well as numerous unstained circular structures in the cytosol, compatible with vacuoles (see Fig. 4a). Immunoblot confirms release of cytochrome c into the cytosol after imatinib treatment for the durations indicated. Original magnification, 40. (c) ATP depletion in NRVMs treated with imatinib (5 M) for 24 h. Shown are the ATP concentration (left) and the ratio of ATP to ADP (right). Data are normalized to 1.0 for vehicle-treated cells. *P < 0.05 versus control. (d) Cleavage of caspase 3 and activity of caspases 3 and 7 after treatment of cardiomyocytes with vehicle or imatinib (5 M) for the time noted (top) or for 26 h (bottom). Data are normalized to 1.0 for vehicle-treated cells. *P < 0.05 versus control. (e) TUNEL assay. NRVMs were treated with vehicle versus imatinib (5 M, top; concentrations indicated below) for 24 h before staining with TUNEL. In the graph, TUNEL-positive cells are expressed as a percentage of the number of total cells, as determined by staining postfixation with propidium iodide (PI). *P < 0.001 versus control; #P < 0.05 versus low-dose imatinib. Original magnification, 40. (f) DNA laddering. NRVMs were treated with vehicle, imatinib (5 M) or, as a positive control, doxorubicin (1 g/ml; Doxo) for 24 h before DNA laddering was determined. The amount of DNA used for the reaction was 2 g (left) or 5 g (right). Full Figure and legend (61K)

Figure 4. Effect of imatinib on characteristics of necrotic cell death. (a,b) Cytosolic vacuolization and lipid droplet accumulation. NRVMs were treated with vehicle or imatinib (5 M) for the durations shown and were then stained with either hematoxylin and eosin (a) or Oil Red-O (b) to identify lipid droplet accumulation. There was pronounced cytosolic vacuolization (a, arrows) and accumulation of lipid droplets (b, red stain). Original magnification, 40. (c) Imatinib induces loss of sarcolemmal integrity as determined by propidium iodide (PI) staining and light microscopy (LM) of live cells. Graph shows the PI-positive cells (those that have lost cell membrane integrity) as a percentage of the total number of cells. Imatinib treatment (5 M) was carried out for 26 h. **P < 0.001 versus control. Original magnification, 20. (d) Imatinib induces cell death in a dose-dependent manner. NRVMs were treated with increasing concentrations of imatinib for 26 h and cellular toxicity was measured by the release of adenylate kinase (AK) into the cell culture medium. *P < 0.05 versus control, **P < 0.001 versus control. Full Figure and legend (63K)

We next examined the mechanisms regulating imatinib-induced cardiomyocyte death. Expression of protein kinase C (PKC), which regulates cardiomyocyte death under conditions of ischemia and oxidant stress^{28, 29}, has been reported to be regulated by c-Abl after oxidant stress¹². We found upregulation and cleavage of PKC in the hearts of mice and in cardiomyocytes exposed to imatinib (Supplementary Fig. 1 online). However, inhibition of PKC with the selective PKC inhibitor peptide V1-1 (ref. 28), or with either of two nonselective PKC inhibitors (GF 109203X and Gö 6983), did not block release of cytochrome c or loss of sarcolemmal integrity in cardiomyocytes in culture (data not shown). Therefore, PKC may be involved in imatinib-induced cardiomyocyte toxicity at a late stage after the cell is committed to die (as has been previously proposed²⁹), but it is not necessary for imatinib-induced cardiomyocyte death.

We next investigated what the PKC-independent pathway mediating death might be. Because of the pronounced accumulation of membranous debris within, and dilation of, the sarcoplasmic and endoplasmic reticulum in the hearts of imatinib-treated humans and mice, we tested whether the ER stress response pathway^{30, 31, 32} was recruited. We found significant activation by imatinib of the PERK to eIF2 arm of the stress response in the hearts of imatinib-treated mice, as assessed by an increase in phosphorylation of eIF2 (Fig. 5a). This arm of the stress response was also activated in cells in culture, but less markedly than in vivo (Fig. 5a).

Figure 5. Signaling pathways regulating imatinib-induced cell death. (a) Imatinib activates the ER stress response in vivo and in vitro. Top left, immunoblot of lysates of hearts from two vehicle-treated and two imatinib-treated mice (50 mg/kg/d for 3 weeks or 100 mg/d for 6 weeks) with a phosphorylation-specific antibody that recognizes activated eIF2. Top right, phosphorylation of eIF2 in vivo normalized to GAPDH as a loading control (n = 4). Bottom, phosphorylated eIF2 in lysates of cardiomyocytes in culture treated with imatinib (5 M) for 6 or 26 h. *P < 0.05 versus control. (b) Activation of IRE1 by imatinib. Cardiomyocytes were treated with imatinib (5 M) or thapsigargin (1 M; Thapsi) with or without the proteasome inhibitor MG-132 (1 M) for the indicated durations. Cell lysates were then immunoblotted for spliced XBP-1. (c) The inhibitor of eIF2 dephosphorylation, salubrinal (Sal), blocks imatinib (Imat)-induced collapse of the mitochondrial potential and cell death. Top, increase in phosphorylated eIF2 in response to treatment with salubrinal. Middle, JC-1 ratio. Bottom, percentage of PI-positive cells. Cells were treated with imatinib (5 M) or vehicle for either 26 h (top and bottom) or 24 h (middle). Salubrinal (20 M) was added 6 h after starting the imatinib treatment. Cells were then collected for imunoblotting (top) or for staining with JC-1 (middle) or PI (bottom). In c and f, PI-positive cells are expressed as a percentage of the number of total cells. *P < 0.001 versus control, #P < 0.001 versus imatinib. (d) Activation of JNK pathway by imatinib. Top, NRVMs were treated with imatinib (5 M) for the durations indicated and then lysates were immunoblotted for phosphorylated JNK. Bottom, cells were treated with imatinib (5 M) for 26 h. Salubrinal (Sal, 20 M) was added into the cell culture medium 6 h after imatinib. (e,f) Inhibition of JNK activity protects NRVMs from imatinib-induced collapse of mitochondrial potential and necrotic death. Cells were pretreated with the cell-permeant JNK peptide inhibitor for 1 h, and were then exposed to vehicle or imatinib for 24 h (e) or 26 h (f) before staining with JC-1 (e) or PI (f). In e, *P < 0.001 versus control, #P < 0.001 versus imatinib. In f, *P < 0.001 versus control, #P < 0.01 versus imatinib. (g) Imatinib induces Bax translocation to, and multimerization at, mitochondria. Imatinib- or vehicle-treated NRVMs were subjected to subcellular fractionation to separate the mitochondria-rich heavy membrane fraction (HM). Top, immunoblot showing Bax translocation to mitochondria after treatment with imatinib for the indicated durations. Bottom, immunoblot showing crosslinked BAX multimers. (h) Imatinib-induced cytochrome c release and cardiomyocyte death are mediated by inhibition of c-Abl. Top left, immunoblot with antibody to c-Abl of lysates from control cells (-) or from cells transduced with either wild-type c-Abl (WT) or c-Abl(T315I). Bottom left, NRVMs transduced with wild-type c-Abl or c-Abl(T315I) were treated with imatinib or vehicle (control) for 24 h, and then fixed and stained for cytochrome c. Right, NRVMs transduced with wild-type c-Abl or c-Abl(T315I) were treated with imatinib or vehicle (control) for 26 h, and cellular toxicity was measured by release of adenylate kinase (AK) into the medium. **P < 0.001 versus vehicle, *P < 0.05 versus vehicle, #P < 0.02 versus wild-type c-Abl plus imatinib. Original magnification, 40. Full Figure and legend (80K)

Phosphorylation of eIF2 attenuates protein translation in general but leads to increased translation of specific mRNAs, responses that are thought to be cytoprotective³¹. We therefore examined whether the other principal arm of the ER stress response, IRE1, was activated. IRE1 is both a protein kinase and an endoribonuclease³³. When IRE1 is activated, its endoribonuclease activity excises an intron from the mRNA of the transcription factor XBP-1, resulting in the conversion of a 267-residue transcriptionally inactive protein, XBP-1u (for unspliced), to a transcriptionally active 371-residue protein, XBP-1s (for spliced)³⁴. XBP-1s then induces transcription of one or more proteins involved in ER-mediated protein degradation³⁵. We found clear-cut activation of the IRE1 arm, as assessed by increased expression of XBP-1s (Fig. 5b). Confirming that the protein identified was XBP-1s, treatment with the proteasome inhibitor MG-132 produced the characteristic reduction in the amount of XBP-1s^{34, 35} (Fig. 5b). Notably, the imatinib-induced increase in XBP-1s was equivalent to that induced by treatment of the cells with the potent ER stressor thapsigargin (Fig. 5b).

To determine whether the increase in ER stress induced by imatinib could be the mechanism triggering cardiomyocyte death, we tested the effects of salubrinal, a small-molecule inhibitor of the dephosphorylation of eIF2 that inhibits cell death selectively induced by ER stressors by maintaining inhibition of the translational control point of the ER stress response³¹. Salubrinal increased phosphorylation of eIF2 and rendered cardiomyocytes resistant to imatinib-induced collapse of the mitochondrial membrane potential, as determined by JC-1 (Fig. 5c), and cell death, as assessed by propidium iodide staining (Fig. 5c). These data are consistent with the idea that the ER stress response is a crucial component of imatinib-induced cell death.

We investigated how the ER stress response might be inducing cardiomyocyte death. As noted above, IRE1-induced expression of XBP-1s is thought to be protective. If the inducing stress is not relieved, however, IRE1 can also signal cell death by activation of the JNK pathway, culminating in mitochondria-dependent cell death^{30, 36, 37}. We therefore examined JNK activation and found marked activation of JNKs both in cultured cardiomyocytes (Fig. 5d) and in the hearts of imatinib-treated mice (Supplementary Fig. 2 online). Salubrinal reduced imatinib-induced JNK activation (Fig. 5d), suggesting that ER stress was largely responsible for JNK activation. Treatment of cells with a selective inhibitor of JNK activity, the JNK inhibitor peptide³⁸, significantly reduced imatinib-induced collapse of the mitochondrial membrane potential, as assessed by JC-1 staining (Fig. 5e). In addition, JNK inhibition significantly reduced imatinib-induced cell death, as determined by prevention of the loss of sarcolemmal integrity (Fig. 5f). These data support the notion that JNKs have a key role in an imatinib-induced process that culminates in collapse of the mitochondrial membrane potential and necrotic cardiomyocyte death. Inhibition of JNKs had no effect on triggering of the ER stress response by imatinib, as determined by eIF2 phosphorylation, consistent with the idea that JNK activation is downstream of the ER stress response (Supplementary Fig. 3 online).

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The effects on cellular energetics were profound, as the ATP concentration dropped by 65% in the cardiomyocytes. In comparison, treatment of cardiomyocytes with another cardiotoxic chemotherapeutic agent, Herceptin, produces only a 35% decline in ATP⁵. Although a 65% reduction in ATP concentration within an individual cell would probably not compromise many cellular processes, an overall reduction of 65% probably reflects a situation in which there are cells with severe depletion of ATP and others with normal or near-normal levels. Thus, the decline in ATP concentration observed might have significant deleterious consequences on various cellular processes in individual cells. In addition, this energy run-down may largely explain why it was difficult to classify the type of cell death induced by imatinib in cultured cardiomyocytes, which included features of both apoptotic and necrotic cell death. On one hand, there were significant increases in TUNEL-positive cells, but activation of caspases was limited, DNA laddering was not significantly increased and nuclear condensation was not evident at times when injury to the cells was advanced. On the other hand, we observed pronounced cytoplasmic vacuolization and loss of plasma membrane integrity, which are both more characteristic of necrotic death²⁶. Because apoptosis is an energy-requiring process, it is possible that the energy rundown induced by imatinib prevented the apoptotic program from being carried out despite the marked release of cytochrome c, which should have, in energy-replete cells, triggered the intrinsic pathway^{26, 27}.

Although alterations in mitochondrial function seem to be the ultimate cause of imatinib-induced toxicity, our data suggest that activation of the ER stress response, acting through JNKs, occurs upstream of the mitochondrial defects. Imatinib strongly recruited the ER stress response because phosphorylation of eIF2 (a marker of PERK activation) and splicing of XBP-1 (a marker of IRE1 activation) were significantly increased in the hearts of treated mice and in cardiomyocytes in culture^{30, 31, 32, 33}. Imatinib also triggers ER stress in CML cells expressing Bcr-Abl⁴¹. Although this observation, like our data, supports the concept that Abl can protect against ER stress, this is not a universal phenomenon, as c-Abl-deficient mouse embryo fibroblasts are protected against ER stress⁴². This difference probably reflects either a 'dosage' effect (partial inhibition with imatinib versus complete deletion) or the different function of c-Abl in different cells, as noted above^{8, 9, 10, 11, 12, 13, 14, 15}. The ER stress response is, at least in the initial stages, cytoprotective, because cells with a deletion of eIF2 are more susceptible to ER stress, and overexpression of eIF2 or prevention of eIF2 dephosphorylation with salubrinal are protective^{26, 27, 30, 31}. If the inciting stressor is not removed and ER stress is prolonged, however, activation of cell-death pathways ensues³⁰.

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Imatinib mesylate (Gleevec, Novartis) capsules were obtained from Tufts–New England Medical Center pharmacy. The 100-mg capsules were dissolved in distilled water and nonsoluble material was removed by repeated centrifugation at 2,500g to yield highly purified material¹⁸. Antibodies to caspase-12, Bax, PKC and cytochrome c were from BD Biosciences. Antibody to Omi was from Biovision. Antibodies to cleaved caspase 3 and phosphorylated eIF2 were from Cell Signaling Technology. The general caspase inhibitor Z-VAD-fmk and antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from R&D Systems, and antibody to vinculin was from Sigma. Antibody specific for phosphorylated JNK was from Promega. Antibody to HSP-60 was from Stressgen Bioreagents. The Bax-inhibiting peptide, GF 109203x, Gö 6983, JNK inhibitor peptide and salubrinal were from Calbiochem. The PKC-inhibiting peptide (V1-1) was from Kai Pharmaceuticals.

We homogenized mouse hearts in lysis buffer consisting of 10 mM Tris (pH 7.4), 250 mM sucrose, 1 mM EDTA and protease inhibitors. We centrifuged the homogenate for 5 min at 1,000g, removed the supernatant and further centrifuged for 15 min at 10,000g to pellet the mitochondria. We washed the pellet twice with buffer consisting of 10 mM Tris (pH 7.4) and 250 mM sucrose, and resuspended the pellet in buffer consisting of 10 mM Tris (pH 7.4), 120 mM KCl and 5 mM KH₂PO₄. After 2 h of incubation on ice, we loaded equal amounts of protein into cuvettes and incubated them at 22 °C for 5 min in the presence of 5 mM sodium succinate and 2 M rotenone. We determined the mitochondrial pore opening by measuring the mitochondrial swelling induced by CaCl₂ (250 M) by detecting light scattering at 520 nm.

We cloned both wild-type and T315I mutant c-ABL Ib⁴⁰, in which Thr315 is mutated to isoleucine, into a retroviral expression vector, MIGR1, containing a reporter gene encoding enhanced green fluorescent protein (eGFP). We sequenced the final products to verify the presence or absence of the Thr315 Ile mutation. We cotransfected 293T cells with retroviral vector and the amphotrophic single-genome packaging construct pKat2ampac to produce replication-defective amphotrophic viral stocks. We determined viral titers by transduction of Rat-1 cells, followed by flow cytometric detection of GFP. We transduced neonatal rat ventricular myocytes (NRVMs) with the viruses for 4 h immediately after their isolation, and the experiments were done 2 d after transduction.

We incubated imatinib-treated myocytes at 37 °C in suspension with F-10 media containing 10 g/ml JC-1 (Molecular Probes) for 10 min, washed them twice with culture media to remove unbound dye, and subsequently subjected them to fluorescence microscopy. Mitochondria in which the mitocondrial membrane potential is maintained accumulate so-called J-aggregates that fluoresce red, whereas those with a collapsed membrane potential fluoresce green. We calculated the mitochondrial membrane potential for single cells by dividing the intensity of the 590 nm (red) image with its corresponding 525 nm (green) image. We incubated MitoTracker Red (chloromethyl-X-rosamine, 200 nM) with cells for 10 min. We then washed, fixed and analyzed the cells by fluorescence microscopy.

Determination of ATP and the ADP/ATP ratio was done according to manufacturers' instructions (Molecular Probes and Bio-Vision, respectively). For propidium iodide staining, we added medium containing propidium iodide (0.5 g/ml) for 5 min and visualized the cells immediately by fluorescence and light microscopy. Release of adenylate kinase was measured in culture supernatants by a bioluminescent ToxiLight kit (Cambrex) according to the manufacturer's instructions.

We fixed the specimens in Trump fixative (4% formaldehyde and 1% glutaraldehyde in phosphate buffer, pH 7.2) transferred into cacodylate buffer, postfixed them in 1% osmium tetroxide in cacodylate buffer for 3 h at 20 °C, and stained them en bloc with 5% aqueous uranyl acetate. We further dehydrated the samples in a graded ethanol series and embedded them in Epon-812. We stained thick sections (1 m) with toluidine blue. We cut thin sections at 50–70 nm, stained them with uranyl acetate and lead citrate, and photographed them with a Philips EM 201 transmission electron microscope.

Cells were collected and DNA was extracted using a kit from Roche. We incubated 2 g of DNA in 10 l of buffer consisting of 50 mM Tris (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol, 50 g/ml BSA, 10 units of Klenow enzyme and 0.5 Ci of [-³²P]dCTP for 10 min at 22 °C. The reaction was stopped by adding 2 l of gel loading buffer and the samples were then loaded onto a 1.8% agarose gel.

Differences between data groups were evaluated for significance using Student t-test of unpaired data or one-way analysis of variance and Bonferroni post-test. Repeated measures analysis of variance was used for multivariate analysis. All experiments were repeated at least three times and the data are presented as the mean s.e.m. unless noted otherwise.

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Competing interests statement:  The authors declare no competing financial interests.