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TOXICOLOGY
Departments of Pharmacology and Toxicology (K.E.M., Y.J.K.) and Medicine (Y.J., W.F., Y.J.K.), University of Louisville School of Medicine, Louisville, Kentucky
Received June 2, 2006; accepted August 18, 2006.
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Abstract |
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). Prolonged hypertrophy is a common endpoint of many cardiovascular diseases and is associated with arrhythmia, sudden death, and heart failure (Ho et al., 1993; Lloyd-Jones et al., 2002). The molecular mechanisms that regulate cardiac myocyte hypertrophy have attracted a great deal of attention, and numerous signal transduction pathways have been implicated in this process (Frey and Olson, 2003). One of these pathways involves the calcium/calmodulin-dependent protein phosphatase, calcineurin.
Calcineurin, a ubiquitously expressed serine/threonine phosphatase, is activated by sustained elevations in intracellular calcium. Activation of calcineurin has been shown to be both necessary and sufficient to induce cardiac hypertrophy in vivo and in vitro (Molkentin et al., 1998). Activated calcineurin facilitates the dephosphorylation of nuclear factor of activated T cells (NFAT), the primary downstream transcriptional effector of calcineurin (Crabtree and Olson, 2002). Calcineurin-mediated dephosphorylation of NFAT leads to the rapid nuclear import of NFAT, induction of fetal cardiac genes, and subsequent hypertrophic response (Molkentin et al., 1998; Crabtree and Olson, 2002). Calcineurin has been shown to be involved in the hypertrophic response of cultured neonatal cardiomyocytes to agonists such as phenylephrine and angiotensin II and has been shown more recently to be activated following treatment with the antineoplastic agent doxorubicin (DOX) (Molkentin et al., 1998; Kalivendi et al., 2005).
DOX is a valuable component of multiple chemotherapeutic regimens; however, severe cardiotoxicity is a major limiting factor that compromises its clinical use (Ferrans, 1978). In vivo, DOX treatment has been shown to result in cardiac hypertrophy (Sun et al., 2001). In vitro, DOX treatment induced cardiomyocyte hypertrophy in primary neonatal rat cardiomyocytes as measured by an increase in cell volume and protein content per cell (Tu et al., 2002). The molecular mechanisms involved in this process have not been thoroughly investigated.
The present study was undertaken to examine the involvement of calcineurin activation in DOX-induced cardiac myocyte hypertrophy. Using the H9c2 rat cardiac-derived cell line and the pharmacologic inhibitors of calcineurin, cyclosporine A (CsA) and FK506, we observed that, although calcineurin is activated by DOX treatment, it is not necessary for DOX-induced cellular hypertrophy. Further investigation into the possible signaling pathways involved in DOX-induced cellular hypertrophy revealed a critical role for the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway.
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Materials and Methods |
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Cell Culture and Treatment. Embryonic rat heart-derived cell line H9c2, obtained from the American Type Culture Collection (Manassas, VA), were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), L-glutamine (4 mM), 1.5 g/l sodium bicarbonate, penicillin (100 U/ml), and streptomycin (100 µg/ml). The cells were maintained at 37°C under a water-saturated atmosphere of 95% ambient air and 5% CO2. Stock cultures were passaged at 2- to 3-day intervals. Cells were seeded at a density of 6.5 x 105 cells/dish in 100-mm dishes for the calcineurin phosphatase assay and immunoblot analysis and 8.5 x 104 cells/dish in 35-mm dishes for measurements of protein content per cell and cell size. The cells were cultured for 24 h before each experiment in DMEM containing 10% FBS.
For experiments involving CsA, FK506, or LY294002, H9c2 cells were pretreated for 2 h with CsA, FK506, or LY294002 before the addition of DOX. Cells were incubated with DOX at 37°C and 5% CO2 in DMEM containing 10% FBS for 2 h. Following the 2-h treatment, DOX and medium were removed from the cells, and fresh medium and CsA, FK506, or LY294002 were added back to the cells where necessary for an additional 6, 22, or 46 h.
Measurement of Protein Content Per Cell. Cells in 35-mm dishes were collected by trypsinization with trypsin-EDTA (Invitrogen) and washed twice in ice-cold phosphate-buffered saline (PBS). Cells were then collected via centrifugation and lysed with 15 µl of cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM 
-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) on ice for 30 min. The suspension was centrifuged at 14,000g for 10 min at 4°C, and the supernatant was collected. Protein concentration in total cell lysate was measured using the Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin (BSA) as standard. Protein content per cell was determined by dividing the total amount of protein by the cell number, which was determined using a hemocytometer.
Measurement of Cell Size. Adherent cells in 35-mm dishes were detached via trypsinization and images of rounded cells were acquired using a Nikon TE2000-S microscope with attached digital camera and 20x lens (Nikon, Tokyo, Japan). Measurements of cell diameters were made using Microsoft Office Document Imaging software (Microsoft, Redmond, WA), and cell volume was calculated using the equation for the volume of a sphere (4/3 x 
x radius3). The diameter of individual cells was measured, and 100 cells per experimental group were measured randomly.
Calcineurin Phosphatase Activity Assay. Calcineurin phosphatase activity was determined as described previously (Fruman et al., 1996). In brief, the calcineurin-specific substrate RII was phosphorylated in vitro with 250 U of reconstituted protein kinase A catalytic subunit, 50 mM ATP, 50 µCi of [
-32P]ATP, 0.15 mM RII peptide, and 500 µl of 2x kinase reaction buffer (40 mM MOPS, pH 7.0, 4 mM MgCl2, 0.1 mM CaCl2, 0.4 mM EDTA, 0.8 mM EGTA, 0.5 mM DTT, and 0.1 mg/ml BSA). Following treatment, H9c2 cells were pelleted by centrifugation, washed twice with ice-cold PBS, and lysed in 50 µl of hypotonic lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 1 mM EDTA, 0.1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail) via three cycles of freeze-thawing in liquid nitrogen and a 30°C water bath. The suspension was centrifuged at 14,000g for 10 min at 4°C, and the supernatant was collected. The protein concentration of each sample was then determined using the Bradford assay, and the concentration of each sample was adjusted to 0.5 to 1 mg/ml with lysis buffer. Calcineurin activity in each sample was determined by incubating equal parts reaction assay buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM DTT, 0.1 mg/ml BSA, 0.5 mM CaCl2, 100 nM calmodulin, and 1 µM okadaic acid), diluted sample protein, and labeled RII peptide at 30°C for 10 min. The reaction was stopped by the addition of 500 µl of 0.1 M potassium phosphate buffer in 5% trichloroacetic acid. To determine the phosphate released in each sample, reactions were then added to Micro Bio-Spin chromatography columns (Bio-Rad) containing Dowex AG 50W-X8 ion exchange resin (Bio-Rad) prepared as described previously (Fruman et al., 1996). Flow-through from each column was then added to 5 ml of scintillation fluid, and the released phosphate was measured in a scintillation counter. The amount of protein was adjusted so that less than 25% of the substrate was consumed during the reaction period. Duplicate counts per minute values from the phosphatase assay were averaged, and the resulting value was adjusted by subtracting the counts per minute in blanks lacking cell lysate. This value was divided by the specific activity of the substrate to give picomoles of phosphate released. The resulting value was divided by the reaction time and the amount of protein to give final values expressed as picomoles of phosphate per minute per milligram of protein.
Adenovirus Propagation and Infection. Adenoviral NFAT-luciferase reporter (AdNFAT-luc) was a generous gift from Dr. Jeffery D. Molkentin (University of Cincinnati, OH) and was described previously (Wilkins et al., 2004). AdNFAT-luc was propagated in human embryonic kidney 293 cells and purified by the ViraBind Adenovirus Purification Kit (Cell Biolabs, Inc., San Diego, CA). H9c2 cells were infected at a multiplicity of infection of 100 plaque-forming units per cell in PBS supplemented with 2% FBS for 2 h at 37°C and 5% CO2, followed by the addition of fresh medium containing FBS for an additional 22 h. The medium was replaced 24 h after infection with DMEM containing 10% FBS for an additional 24 h before treatment. Under these conditions, approximately 98% of cells were infected as assessed by infection with the same titer of Ad-GFP.
Luciferase Enzymatic Assay. Luciferase enzymatic activity in the cell extracts was measured using the Luciferase Assay System (Promega, Madison, WI) according to the supplier's instructions. The light intensity was measured with a luminometer (LMax; Molecular Devices, Sunnyvale, CA) over 10 s and expressed as relative light units over 10 s/µg protein.
Immunoblot Analysis. Cell extracts were prepared as described above using cell lysis buffer. Protein concentration in total cell lysate was measured using the Bradford assay with BSA as standard. Cell lysates (50 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were subsequently incubated overnight at 4°C in primary antibody (anti-phospho-Akt, anti-Akt, or anti-GAPDH antibodies) and then with secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The membranes were then developed with the enhanced chemiluminescence kit (GE Healthcare, Piscataway, NJ). Densitometry was performed using ImageQuant 5.2 software (GE Healthcare).
Statistical Analysis. Data are presented as mean ± S.D. Data were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's method for multiple comparison. Experiments involving a two by two factorial experimental design were analyzed by two-way ANOVA. After a significant interaction was detected by the two-way ANOVA, the significance of the main effects was further determined. The level of significance was considered at P < 0.05.
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). Thus we examined the effect of DOX treatment on calcineurin activity in H9c2 cells. The total calcineurin enzymatic activity was measured in H9c2 cells exposed to increasing concentrations of DOX (1, 2, and 5 µM) for 2 h followed by 6 h of recovery (8 h total) in fresh DMEM containing 10% FBS. DOX treatment induced a dose-dependent increase in calcineurin activity as measured by the calcineurin phosphatase activity assay (Fig. 1C).
Cyclosporine A and FK506 Inhibit DOX-Induced Calcineurin-NFAT Signaling. Analysis of NFAT transcriptional activity is a functional assessment of calcineurin activity and an alternative method of assessing calcineurin activation (Wilkins et al., 2004). To more carefully evaluate the activation of calcineurin and the calcineurin-NFAT signaling pathway by DOX, H9c2 rat cardiac cells were infected with AdNFAT-luc. Treatment of AdNFAT-luc infected H9c2 cells with 1 µM DOX for 2 h followed by 22 h of recovery (24 h total) induced a significant increase in NFAT reporter activity (Fig. 2).
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Cyclosporine A and FK506 Do Not Inhibit DOX-Induced Cellular Hypertrophy. If calcineurin activation mediates DOX-induced hypertrophy in H9c2 cells, CsA and FK506 would inhibit DOX-induced hypertrophy. We exposed H9c2 cells to 100 nM CsA or 1 µM FK506 for 2 h prior to and throughout treatment with 1 µM DOX. After the 2-h DOX treatment, the cells were allowed to recover in fresh medium containing the inhibitors for an additional 46 h (48 h total). Although the cell volume and protein content per cell following treatment with 1 µM DOX were significantly increased, treatment with CsA or FK506 did not affect the DOX-induced hypertrophic response in H9c2 cells (Fig. 3, A and B). Collectively, these results indicate that, although calcineurin is activated by DOX treatment, it is not necessary for DOXinduced hypertrophy in H9c2 cells.
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The PI3K-Akt Signaling Pathway Is Involved in DOX-Induced Cellular Hypertrophy. If calcineurin activation is not involved in DOX-induced hypertrophy in H9c2 cells, what is (are) the signaling pathway(s) that are involved in DOX-induced hypertrophy? DOX has been shown to activate/phosphorylate the serine/threonine protein kinase Akt (Deres et al., 2005; Li et al., 2005), and cardiac-specific expression of a constitutively active form of Akt has been shown to induce significant cardiac hypertrophy in transgenic mice (Matsui et al., 2002; Shioi et al., 2002). Therefore, we examined the possibility of Akt involvement in DOX-induced hypertrophy. H9c2 cells were exposed to 1 µM DOX for 5, 15, 30, or 60 min or for 2 h followed by removal of DOX for an additional 1 (180 min) or 4 (360 min) h. Akt phosphorylation on serine 473 (Ser473) was subsequently evaluated. Ser473 phosphorylation of Akt was detected at 180 min after the addition of DOX, and phosphorylation was maintained up to 360 min, whereas total Akt levels were unaltered (Fig. 4, A and B). As a positive control, exposure of H9c2 cells to IGF-1 for 5 min resulted in rapid phosphorylation of Ser473 of Akt (Fig. 4, A and B).
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The primary upstream regulator of Akt is PI3K. Therefore, we then examined the possibility of PI3K involvement in DOX-induced hypertrophy. H9c2 cells were exposed to increasing concentrations of the specific PI3K inhibitor LY294002 for 2 h prior to and throughout treatment with 1 µM DOX. After the 2-h DOX treatment, the cells were allowed to recover in fresh medium containing LY294002 for an additional 46 h (48 h total). Measurements of protein content per cell showed that LY294002 could inhibit the DOX-induced increase in protein content per cell in a dose-dependent manner and that 20 µM LY294002 can completely inhibit the DOX-induced increase in protein content per cell (Fig. 5). In addition, not only was the DOX-induced increase in protein content per cell inhibited in the presence of 20 µM LY294002 but also the DOX-induced increase in cell volume was significantly inhibited at this concentration of PI3K inhibitor (Fig. 6, A and B). It should also be noted that treatment of H9c2 cells with 20 µM LY294002 alone was not cytotoxic to cells (data not shown). These results indicate that the PI3K-Akt signaling pathway is critically involved in DOX-induced cellular hypertrophy.
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Discussion |
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H9c2 cells are commonly used as an in vitro model for studying the cellular mechanisms and signaling pathways involved in DOX-induced cardiotoxicity and have been used previously as an experimental model of hydrogen peroxide (H2O2)-induced hypertrophy (Chen et al., 2000). The H9c2 cell line is a clonal cardiomyocyte cell line derived from embryonic rat ventricles. These cells maintain many molecular markers of cardiomyocytes and show morphological characteristics of immature embryonic cardiomyocytes, although these cells have dedifferentiated to an extent (Hescheler et al., 1991). Nonetheless, the hypertrophic response of H9c2 cells following H2O2 treatment was found to be similar to that of primary neonatal rat cardiomyocytes that maintain many characteristics of cardiac cells in vivo (Chen et al., 2000). Therefore, these cells were chosen for studying the signaling pathways involved in DOX-induced hypertrophy.
Cardiac hypertrophy is a common endpoint of many cardiovascular diseases and is characterized by an increase in cardiomyocyte size and protein content. Numerous signal transduction pathways have been implicated in the development of cardiac hypertrophy, one of which involves the calcium/calmodulin-dependent protein phosphatase calcineurin. Calcineurin has been shown to be both necessary and sufficient to induce cardiac hypertrophy in vivo and in vitro (Molkentin et al., 1998). Recently, Kalivendi et al. (2005) demonstrated that treatment with the anthracycline quinone DOX activates calcineurin-NFAT signaling in H9c2 rat cardiac cells. However, the effect of this activation on hypertrophy was not investigated. DOX has been shown to induce cardiac hypertrophy in vivo (Sun et al., 2001). DOX treatment of primary neonatal rat cardiomyocytes also results in cardiomyocyte enlargement as measured by increases in cell volume and protein content per cell (Tu et al., 2002). Therefore, in this study, we sought to examine the involvement of calcineurin activation in DOX-induced hypertrophy in H9c2 cells. The results demonstrate that although calcineurin is activated by DOX treatment, it is not necessary for DOX-induced hypertrophy in H9c2 cells.
Although the sufficiency of calcineurin as a transducer of the hypertrophic response is well documented, the necessity of calcineurin in the promotion of cardiomyocyte hypertrophy is less than clear. Although some studies note a necessity of calcineurin signaling for hypertrophy, several studies using a variety of models have shown no effect of CsA or FK506 on the development of cardiac hypertrophy (Luo et al., 1998; Müller et al., 1998; Sussman et al., 1998; Ding et al., 1999; Zhang et al., 1999). Furthermore, additional studies using a familial hypertrophic cardiomyopathy model have observed an exaggeration in the hypertrophic response following treatment with CsA or FK506 (Fatkin et al., 2000; Schmitt et al., 2003). Although the validity of these studies using CsA and FK506 have been called into question due to the nonspecific effects of CsA and FK506, it is more likely that the observed cardiac hypertrophic responses are model-dependent and multifactorial in nature and thereby involve calcineurin-independent signaling pathways. The results obtained in the present study support this notion because inhibition of calcineurin with CsA or FK506 fails to inhibit DOX-induced hypertrophy, whereas specific inhibition of PI3K with LY294002 prevents DOX-induced hypertrophy.
PI3K is a lipid kinase that has been shown to play a crucial role in a multitude of cellular functions, including cell growth, proliferation, and survival (Cantley, 2002). PI3K, which lies downstream of multiple receptor tyrosine kinases and G protein-coupled receptors, catalyzes the addition of a phosphate group to the free 3' position of the inositol ring of phosphoinositides, and the resulting products have been shown to activate signaling pathways involved in hypertrophy (Chen et al., 2001). Activation of PI3K has been observed in vivo in pressure overload hypertrophy, and cardiac-specific expression of constitutively active PI3K in mice resulted in enlarged hearts attributable to an increase in cardiomyocyte size (Naga Prasad et al., 2000; Shioi et al., 2000). Conversely, expression of a dominant-negative mutant of PI3K resulted in smaller hearts and smaller cardiomyocytes compared with normal littermates (Shioi et al., 2000). In vitro, PI3K is activated by H2O2 treatment, and inhibition of PI3K with wortmannin prevents H2O2-induced cardiomyocyte hypertrophy (Tu et al., 2002). Moreover, cardiac-specific expression of a constitutively active form of Akt, a primary downstream effector of PI3K, induces significant cardiac hypertrophy in transgenic mice (Matsui et al., 2002; Shioi et al., 2002). Furthermore, the PI3K-Akt signaling pathway has been implicated in multiple forms of hypertrophy both in vivo and in vitro (Ha et al., 2005; Kenessey and Ojamaa, 2006). Collectively, these data suggest a role for the PI3K-Akt signaling pathway in the development of cardiomyocyte hypertrophy.
Our study reveals a potentially critical role for PI3K in DOX-induced hypertrophy in H9c2 cells. The finding of PI3K involvement in DOX-induced hypertrophy agrees with the observation of PI3K involvement in cardiomyocyte hypertrophy induced by oxidative stress (Chen et al., 2000; Tu et al., 2002). The proposed mechanism for the cardiotoxicity of DOX involves the production of reactive oxygen species/oxidative stress during its intracellular metabolism (Myers et al., 1977; Kang et al., 1996, 1997; Sun et al., 2001). DOX is metabolized by NADH reductase, which is involved in complex I of the mitochondrial respiratory chain. In this reaction, DOX is converted to reduced semiquinone free radical (Davies and Doroshow, 1986). This semiquinone free radical intermediate is highly unstable and rapidly reacts with molecular oxygen to generate superoxide free radical with regeneration of intact DOX (Jung and Reszka, 2001). Furthermore, redox cycling of DOX in the mitochondria generates not only superoxide but also H2O2 and hydroxyl radical (Doroshow and Davies, 1986). Although oxidative stress is implicated in DOX-induced hypertrophy in vitro and in vivo, the role of PI3K/Akt has not been assessed. The data obtained here suggest that oxidative stress induced activation of PI3K/Akt signaling is a potential mechanism involved in DOX-induced hypertrophy.
Although future investigation will further confirm the involvement of the PI3K/Akt signaling pathway in DOX-induced hypertrophy, it would be predicted that other pathways may be involved. In fact, DOX has been shown to activate multiple signaling molecules simultaneously, including the p38 mitogen-activated protein kinase signaling pathway (Kang et al., 2000), which has been implicated in the development of hypertrophy (Zechner et al., 1997; Wang et al., 1998). In addition, our recent studies have shown that DOX treatment can lead to the modification of specific mitochondrial proteins (Merten et al., 2005). The fact that DOX can activate multiple signaling molecules may suggest that a significant amount of cross-talk exists among multiple hypertrophic signaling pathways; therefore, careful consideration of these pathways will be necessary in future evaluations of DOX-induced hypertrophy.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NFAT, nuclear factor of activated T cell; DOX, doxorubicin; CsA, cyclosporine A; FK506, tacrolimus; PI3K, phosphoinositide 3-kinase; IGF-1, insulin-like growth factor-1; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; AdNFAT-luc, adenoviral NFAT-luciferase reporter; ANOVA, analysis of variance.
Address correspondence to: Dr. Y. James Kang, Department of Medicine, University of Louisville School of Medicine, 511 South Floyd Street, MDR 530, Louisville, KY 40202. E-mail: yjkang01{at}louisville.edu
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, significantly different from DOX group.
