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H2O2 regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways
Journal of Molecular and Cellular Cardiology 35 (2003) 615–621 www.elsevier.com/locate/yjmcc
Original Article
H2O2 regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways Susan H. Kwon, David R. Pimentel, Andrea Remondino, Douglas B. Sawyer, Wilson S. Colucci * Myocardial Biology Unit and Cardiovascular Division, Boston University Medical Center, and Boston University School of Medicine, 88 East Newton Street, Boston, MA 02118, USA Received 4 February 2003; accepted 24 February 2003
Abstract Reactive oxygen species (ROS) can act as signaling molecules to stimulate either hypertrophy or apoptosis in cardiac myocytes. We tested the hypothesis that the phenotypic effects of ROS are due to differential, concentration-dependent activation of specific kinase signaling pathways. Adult rat ventricular myocytes were exposed to H2O2 over a broad concentration range (10–1000 µM). Low concentrations of H2O2 (10–30 µM) increased protein synthesis without affecting survival. Higher concentrations of H2O2 (100–200 µM) increased apoptosis (assessed by TUNEL). Still higher concentrations of H2O2 (300–1000 µM) caused both apoptosis and necrosis. A hypertrophic concentration of H2O2 (10 µM) increased the activity of ERK1/2, but not that of JNK, p38 kinase or Akt. An apoptotic concentration of H2O2 (100 µM) activated JNK, p38 kinase and Akt, and further activated ERK1/2. The MEK1/2 inhibitor U0126 prevented the hypertrophic effect of 10 µM H2O2. The apoptotic effect of 100 µM H2O2 was inhibited bya dominant-negative JNK adenovirus, and was potentiated by U0126 or an Akt inhibitor. Thus, the concentration-dependent effects of ROS on myocyte hypertrophy and growth are due, at least in part, to the differential activation of specific kinase signaling pathways that regulate hypertrophy and apoptosis. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: H2O2; Apoptosis; Hypertrophy; Cardiac myocyte; Reactive oxygen species; ERK1/2; JNK; c-Jun; p38 kinase; Akt
1. Introduction Reactive oxygen species (ROS) have been implicated in the pathophysiology of myocardial failure [1–3]. Recent investigations using cardiac myocyte systems in culture show that ROS and oxidative stress can cause multiple changes in cell structure and function that are associated with the failing heart, and which appear to be related to the quantity and type of ROS [4]. For example, in neonatal rat cardiac myocytes (NRVM) a small increase in ROS due to inhibition of superoxide dismutase (SOD) causes hypertrophy, whereas a larger increase due to more complete inhibition of SOD causes apoptosis [5]. Similarly, direct addition of ROS leads to apoptosis in NRVM [6,7], H9c2 cells [8,9], and adult rat ventricular myocytes (ARVM) [10], while hypertrophy has been reported in NRVM and H9c2 cells that survived ROS exposure [9]. Other studies using antioxidants * Corresponding author. Tel.: +1-617-638-8706; fax: +1-617-638-8712. E-mail address: [email protected] (W.S. Colucci). © 2003 Elsevier Science Ltd. All rights reserved. DOI: 10.1016/S0022-2828(03)00084-1
suggest that ROS are involved in mediating the hypertrophic and apoptotic effects of pathophysiologically-relevant stimuli such as mechanical strain [11,12], neurohormonal stimulation [13–15] and inflammatory cytokines [14]. While the source(s) of ROS activated by these pathophysiological stimuli is not yet known, several studies have investigated the mechanism by which ROS activate hypertrophic and apoptotic phenotypes in myocytes. ROS are known to activate ERK1/2, p38 kinase, JNK and Akt in many cell types including cardiac myocytes [16]. The role of these pathways in myocyte hypertrophy and apoptosis remains controversial and incompletely defined. However, collectively several studies support a model in which distinct signaling pathways have differential sensitivity to oxidative stress, and thereby lead to the specific myocyte phenotypes of hypertrophy and apoptosis. For example, activation of ERK1/2 in the absence of JNK, p38 kinase or Akt activation occurs in ARVM in an ROS-dependent manner after a-adrenergic receptor stimulation, leading to myocyte hypertrophy in the absence of apoptosis [17]. Similarly, low-level mechanical strain acti-
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vates ERK1/2, but not JNK, and results in hypertrophy in the absence of apoptosis, also in an ROS-dependent manner [12]. In contrast, in ARVM high concentrations of H2O2 cause apoptosis and JNK activation, and overexpression of a dominant-negative SEK1 construct inhibits both JNK activation and apoptosis [10]. H2O2 activates ERK1/2 and JNK in a concentration-dependent manner in ARVM [18]. Taken together, these studies led to our hypothesis that differential, concentration-dependent activation of kinase cascades determines whether oxidative stress leads to myocyte hypertrophy or apoptotic cell death. Accordingly, the first goal of this study was to determine if H2O2 causes distinct, concentration-dependent phenotypes in ARVM. Finding that low and high concentrations of H2O2 cause hypertrophy and apoptosis, respectively, we next tested whether the concentration-dependent effects of ROS on myocyte phenotype are due to differential activation of distinct kinase signaling pathways. 2. Methods 2.1. Isolation of adult rat cardiac myocytes As previously described [19] ARVM (90–95% purity) were isolated from the hearts of adult (200–220 g) male Sprague-Dawley rats, plated at a non-confluent density of 30–50 cells/mm2 on 100-mm plastic culture dishes (Fisher) or 40 × 22-mm2 glass coverslips (Fisher) precoated with laminin (1 µg/cm2, Becton-Dickinson), and maintained in ACCT medium (DMEM; BSA, 2 mg/ml; L-carnitine, 2 mmol/l; creatinine, 5 mmol/l; taurine, 5 mmol/l; penicillin, 100 IU/ml; streptomycin, 10 µg/ml) for 16 h before drug treatment.
H2O2, ARVM were infected with the dn-JNK or b-galactosidase adenovirus at a MOI of 10. 2.4. Cell viability Cells cultured on six well plates were exposed to H2O2 for 24 h. Methylthiazolyldiphenyl-tetrazolium bromide (MTT; 0.2 mg/ml; Sigma) was added and cells were incubated for 3 h at 37 °C in the dark. DMSO and Sorensen’s glycine buffer (0.1 mol/l glycine, 0.1 mol/l NaCl, pH 10.5 with 0.1 N NaOH) were added, and absorbance (OD) was measured at a wavelength of 570 nm. Cell viability was calculated as the percentage of control OD. 2.5. Total myocyte protein ARVM were washed with a phosphate-buffered solution and harvested in a buffer containing 20 mM Tris-HCl (Sigma), 0.5 mM EDTA (Sigma), 0.3 mM EGTA (Sigma), 1 mM dithiothreitol (Sigma) and 1 µM leupeptin (Sigma). Cells were sonicated, precipitated with 10% TCA and resuspended in 0.4 mol/l NaOH buffer. Aliquots were removed for determination of total cellular protein by the Bradford method using Coomassie Blue (BioRad Laboratories). Cellular protein content was normalized to DNA content, which was measured by fluorometric assay after cell lysis. 2.6. 3H-leucine incorporation Incorporation of 3H-leucine was measured over the final 6 h of H2O2 treatment. The activity of 3H-leucine was determined by scintillation counting of samples harvested and precipitated as noted above. 2.7. Histochemistry
2.2. Drug treatments Myocytes were treated with H2O2 (10–1000 µM, Sigma) for 48 h for hypertrophy and 24 h for apoptosis assays, and for 15–60 min for kinase assays. Inhibitors of MEK1/2 (U0126, 10 µM, Calbiochem), p38 kinase (SB20358, 5 µM, Calbiochem) and Akt (1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, 10 µM, Calbiochem) were added 30 min prior to H2O2. 2.3. Adenoviral constructs The T183A, Y185F JNK1 mutant (dn-JNK) [20] (a kind gift from R. Davis, Worcester, MA) was inserted into pAxCAWt cosmid using the Cos-TPC method. pAxCAWt-dnJNK and a DNA-TPC fragment were transfected into HEK 293 cells using the calcium phosphate method and adenoviral plaques were selected. The plaques were amplified in HEK 293 cells and purified using a double cesium chloride gradient. Using the TCID50 method a viral titer was determined. Similar methods were used for the creation of the b-galactosidase vector. Forty-eight hours prior to addition of
TUNEL staining was performed on cells plated on glass coverslips using a Boehringer Mannheim in situ death detection kit according to the manufacturer’s instructions as previously described [19]. The percentage of TUNEL-positive myocytes (relative to total myocytes) was determined by counting 200 cells per coverslip per condition in randomly chosen fields. A total of 1200 cells from six coverslips were counted for each experimental condition. Annexin V (AnV) with propidium iodide (PI) co-staining was performed using a Molecular Probes Vybrant Apoptosis assay kit according to the manufacturer’s instructions. 2.8. Western blotting Cells were collected in lysis buffer (in mM: 1% Triton X100, 0.5% NP-40, 10 Tris, 1 EDTA, 1 EGTA, 150 NaCl, 0.4 PMSF, 0.2 sodium orthovanadate, 1 µg/µl leupeptin). Protein concentration was determined using Bradford method (BioRad). Equal amounts of total protein were separated by SDS-PAGE on 10% gels, and transferred to Immobilin-P transfer membrane (Amersham), which was probed with
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anti-phospho-ERK1/2 (1:1000, Cell Signaling), antiphospho-JNK (1:1000, Santa Cruz), anti-phospho-c-Jun (1:1000, Cell Signaling), anti-phospho-p38 kinase (1:1000, Cell Signaling) or anti-phospho-Akt (1:1000, Cell Signaling) antibodies. Chemiluminescence was quantified by densitometry (Molecular Analyst, Biorad). 2.9. Statistics All data are presented as mean ± S.E.M. Differences among conditions were determined by ANOVA followed by a paired t-test with the Bonferroni correction for multiple comparisons. P values <0.05 were considered significant.
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3. Results 3.1. Low H2O2 concentrations increase protein synthesis Total myocyte protein and 3H-leucine incorporation were assessed as measures of myocyte hypertrophy. H2O2 at concentrations of 10 or 30 µM increased total protein content by approximately 21–24% (Fig. 1A). At H2O2 concentrations of 100 µM and higher there was a decrease in cell viability associated with cell loss (see below). Likewise, 3H-leucine incorporation was increased by 33–43% at H2O2 concentrations of 10–30 µM (Fig. 1B). 3.2. Cell viability ARVM were exposed to increasing concentrations of H2O2 ranging from 10 to 1000 µM for 24 h and viability was assessed by MTT uptake. There was no decrease in cell viability at H2O2 concentrations of 10 and 30 µM. Cell viability decreased slightly at H2O2 concentrations of 100 µM, and markedly at 300 or 1000 µM (Fig. 2A).
B Fig. 1. Effect of H2O2 on total protein and 3H-leucine incorporation. Myocytes were exposed to H2O2 for 48 h. H2O2 concentrations of 10 and 30 µM increased both total protein (Panel A) and 3H-leucine incorporation (Panel B). At concentrations of 100 µM and higher total protein and 3H-leucine incorporation decreased due to cell loss (see Fig. 2). The data are normalized to DNA content. Shown are the mean from eight to nine experiments. In this and subsequent figures, differences among multiple comparisons were tested by ANOVA followed by paired t-testing with the Bonferroni correction for multiple comparisons (* P < 0.05 vs. control;† P < 0.01 vs. control).
3.3. Higher concentrations of H2O2 stimulate apoptosis H2O2 concentrations of 10–30 µM had no effect on the frequency of apoptotic myocytes as assessed by TUNEL staining. At 100 µM, the frequency of apoptosis increased 4.5-fold (Fig. 2B). AnV (Panel 2C) and PI (Panel 2D) costaining in unfixed cells showed that at an H2O2 concentration of 100 µM AnV staining was increased in the absence of nuclear PI staining, consistent with apoptosis. At an H2O2 concentration of 300 µM there was a marked increase in PI staining, indicating that some of the increase in TUNEL staining at 300 µM H2O2 is due to necrosis. Thus, H2O2stimulated myocyte apoptosis occurs over a relatively narrow concentration range between 30 and 300 µM.
An H2O2 concentration of 10 µM increased the phosphorylation of ERK1/2 2.3-fold with the peak effect at 15 min, but had no consistent effect on p38 kinase or Akt phosphorylation at any time from 5 min to 24 h. There was no increase in c-Jun phosphorylation. In contrast, an H2O2 concentration of 100 µM increased the phosphorylation of p38 kinase, Akt, JNK and c-Jun, and further increased ERK1/2 phosphorylation.
3.5. ERK1/2 mediates hypertrophy at low concentrations of H2O2
3.4. H2O2 concentration-dependent kinase activation H2O2 caused concentration-dependent activation of ERK1/2, JNK, p38 kinase and Akt as assessed by western blotting using antibodies to phosphorylated kinases (Fig. 3).
The MEK1/2 inhibitor U0126 (10 µM) prevented H2O2stimulated (10 µM) hypertrophy (Fig. 4). U0126 prevented H2O2-stimulated activation of ERK1/2, but not the activation of JNK or p38 kinase (data not shown).
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Fig. 2. Concentration-dependent effect of H2O2 on viability and apoptosis in ARVM. Myocytes were exposed to various concentrations of H2O2 for 24 h and viability was assessed by MTT uptake (Panel A). The data are the mean from three experiments. Myocytes were exposed to H2O2 in concentrations ranging from 10 to 1000 µM and apoptosis was assessed by staining for TUNEL (Panel B), AnV (Panel C) or PI (Panel D). TUNEL staining was performed after exposure for 24 h, and AnV and PI staining were performed after exposure for 6 h. Under control conditions 4.6 ± 1% of myocytes were TUNEL-positive, 3.14 ± 2.3% were AnV-positive/PI-negative and 6.5 ± 1.8% were AnV-positive/PI-positive. Exposure to low concentrations of H2O2 (10–30 µM) had no effect on TUNEL, AnV or PI staining. An H2O2 concentration of 100 µM increased the frequencies of TUNEL and AnV-positive/PI-negative staining by 4.5- and 8.8-fold, respectively, without affecting the rate of AnV-positive/PI-positive staining. An H2O2 concentration of 300 µM significantly increased AnV-positive/PI-positive staining consistent with necrosis or other form of cell death. The data are the mean from six (TUNEL) or three (AnV and PI) experiments (* P < 0.05 vs. control; † P < 0.01 vs. control).
3.6. JNK promotes apoptosis at high concentrations of H2O2, whereas Akt and ERK1/2 are anti-apoptotic Apoptosis stimulated by a high concentration of H2O2 (100 µM) was decreased approximately 57% by infection with an adenoviral vector for a dominant-negative JNK (Fig. 5A). In contrast, the MEK1/2 inhibitor U0126 (Fig. 5B) and the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Fig. 5C) each increased H2O2-stimulated apoptosis by approximately 60%. The p38 kinase inhibitor SB20358 had no consistent effect on H2O2-stimulated apoptosis (Fig. 5D). 4. Discussion There are four major new findings of this study. First, H2O2 caused concentration-dependent effects on ARVM phenotype, resulting in hypertrophy at low concentrations
and apoptosis at higher concentrations. Second, these effects on phenotype were associated with concentration-dependent activation of four kinase signaling pathways—ERK1/2, JNK, p38 kinase and Akt. Third, H2O2-stimulated hypertrophy was mediated by ERK1/2, whereas H2O2-stimulated apoptosis was mediated by JNK. Finally, ROS-dependent activation of ERK1/2 and Akt exerted anti-apoptotic actions, which opposed the pro-apoptotic effect of JNK. The concentration-dependent hypertrophic and apoptotic phenotypes induced by H2O2 in ARVM is similar to what we have previously reported in NRVM in response to SOD inhibition or mechanical strain. In neonatal rat cardiac myocytes we found that an increase in ROS due to inhibition of SOD or addition of xanthine oxidase resulted in hypertrophy and apoptosis: hypertrophy occurred at low levels of oxidative stress, whereas apoptosis occurred at higher levels of oxidative stress [5,12]. Likewise, ROS have also been implicated in mediating both hypertrophy and apoptosis in adult
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Fig. 3. Concentration-dependent effects of H2O2 on kinase activity. Myocytes were exposed to H2O2 concentrations of 10 or 100 µM for 15 min, and the phosphorylation of ERK1/2, JNK, p38 kinase and Akt was determined by western blotting. The phosphorylation of c-Jun was determined at 60 min. H2O2 at a concentration of 10 µM, increased the activation of ERK1/2 (Panel A), but not JNK (Panel B), c-Jun (Panel C), p38 kinase (Panel D) or Akt (Panel E). At an H2O2 concentration of 100 µM, there was activation of p38 kinase, JNK, c-Jun and Akt, and more intense activation of ERK1/2 (* P < 0.05 vs. control; † P < 0.01 vs. control).
effects of a-adrenergic receptor agonists and endothelin in ARVM. The magnitude of the hypertrophic effect of 10–30 µM H2O2 was less than that of serum, but similar to what we have previously observed for a-adrenergic stimulation in ARVM [13]. Aoki et al. [10] showed that ROS can cause apoptosis in ARVM. In the present study we found that H2O2 can cause both hypertrophy and apoptosis in a concentrationdependent manner in ARVM. Taken together with the prior studies in NRVM, we conclude that the effects of ROS on myocyte phenotype are determined, at least in part, by the concentration of ROS.
Fig. 4. Prevention of H2O2-stimulated hypertrophy by the MEK1/2 inhibitor U0126. Myocytes pretreated with U0126 (10 µM; 30 min) were exposed to a hypertrophic concentration of H2O2 (10 µM) for 24 h. Data depict the mean total cellular protein content from nine experiments (* P < 0.05 vs. control; † P < 0.05 vs. H2O2).
cardiac myocytes. Amin et al. [13] and Tanaka et al. [15] have shown that ROS are involved in mediating the hypertrophic
Although high concentrations of ROS can cause toxic cell injury, there is now evidence that lower, sub-toxic concentrations of ROS can act as intracellular signaling molecules [4,21]. ROS activate a variety of kinase signaling pathways that have been implicated in the regulation of myocyte hypertrophy and/or apoptosis, including ERK1/2, JNK, p38 kinase and Akt. We found that the pattern of kinases activated was related to the concentration of H2O2, a finding that is generally similar to that of Wei et al. [18]. At low concentrations,
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B
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D Fig. 5. Effects of kinase inhibitors on H2O2-stimulated apoptosis. Panel A. Myocytes were infected with an adenoviral vector for a dominant-negative mutant of JNK1 for 24 h prior to exposure to H2O2 for an additional 24 h. Apoptosis was assessed by TUNEL staining. Data are the mean from three experiments (* P < 0.01 vs. control; † P < 0.05 vs. H2O2). Panel B. Pretreatment with the MEK1/2 inhibitor U0126 (10 µM; 30 min). Data are the mean from four experiments (* P < 0.01 vs. control; † P < 0.01 vs. H2O2). Panel C. Pretreatment with the Akt inhibitor 1L-6-hydroxymethylchiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (10 µM; 30 min). Data are the mean from four experiments (* P < 0.01 vs. control; † P < 0.01 vs. H2O2). Panel D. Pretreatment with the p38 kinase inhibitor SB20358 (5 µM; 30 min). Data are the mean from five experiments (* P = 0.0083 vs. control).
H2O2 activated primarily ERK1/2, but not JNK, p38 kinase or Akt. With higher concentrations, we found increased activation of ERK1/2, along with activation of JNK, p38 kinase and Akt, and increased c-Jun phosphorylation. The hypertrophic effect of 10 µM H2O2 was markedly decreased by the MEK1/2, but not the JNK inhibitor. We found that the Akt inhibitor also attenuated hypertrophy. However, given the lack of consistent activation of Akt by 10 µM H2O2, it is possible that this reflects non-selective effect of the Akt inhibitor on ERK1/2 signaling. Therefore, although the data support an important role for ERK1/2 in mediating the hypertrophic effect of 10 µM H2O2, we can neither implicate nor exclude a role for Akt with these data. The apoptotic effect of 100 µM H2O2 was inhibited by infection with an adenovirus carrying dominant-negative JNK. These findings are consistent with the demonstration by Aoki et al. [10] that H2O2-stimulated apoptosis in ARVM is prevented by overexpression of a dominant-negative SEK1. Likewise, JNK has been implicated in mediating ROSstimulated apoptosis in the H9c2 muscle cell line [8,22]. However, there is also evidence that JNK can mediate antiapoptotic effects in neonatal rat cardiac myocytes [23,24] and embryonic stem cell-derived cardiac myocytes [25]. These divergent results may be due to differences in kinase signaling pathways related to developmental stage or culture conditions. We did not assess hypertrophy at higher concentrations of H2O2 that caused apoptosis. However, Chen et al. [9] have suggested that apoptotic concentrations of H2O2 may also be associated with hypertrophy of the surviving cells. Like other investigators, we found that ERK1/2 or Akt pathways activated at high concentrations of H2O2 play a protective role against H2O2-induced apoptosis [6,26]. These findings suggest that the regulation of apoptosis by ROS involves multiple kinase signaling pathways, with the net effect reflecting a balance between the pro-apoptotic effect of JNK and the anti-apoptotic actions of ERK1/2 and Akt. Taken together, these data suggest that the level of ROS is an important determinant of myocyte phenotype via the differential activation and interplay of multiple kinase signaling pathways. There is increasing evidence that a variety of stimuli including mechanical strain [11,12], neurohormonal stimulation [13–15] and inflammatory cytokines [14] can increase the level of ROS in cardiac myocytes. The present findings help to elucidate the complex role that ROS play in mediating myocyte phenotype and provide a mechanism by which a stimulus may exert diverse effects on myocyte hypertrophy and apoptosis. Acknowledgements We are grateful to Paul O. Kwon for his technical expertise and helpful discussions. We also thank Jing Wang and Xinxin Guo for their excellent technical assistance. This work was supported by NIH grants HL61639 and HL20612 (W.S.C.), HL03878 (D.B.S.), an NIH Cardiovascular Scientist Training Grant HL07224 (S.H.K. and D.R.P.); and a grant from the Swiss National Science Foundation (A.R.).
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Original Article
H2O2 regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways Susan H. Kwon, David R. Pimentel, Andrea Remondino, Douglas B. Sawyer, Wilson S. Colucci * Myocardial Biology Unit and Cardiovascular Division, Boston University Medical Center, and Boston University School of Medicine, 88 East Newton Street, Boston, MA 02118, USA Received 4 February 2003; accepted 24 February 2003
Abstract Reactive oxygen species (ROS) can act as signaling molecules to stimulate either hypertrophy or apoptosis in cardiac myocytes. We tested the hypothesis that the phenotypic effects of ROS are due to differential, concentration-dependent activation of specific kinase signaling pathways. Adult rat ventricular myocytes were exposed to H2O2 over a broad concentration range (10–1000 µM). Low concentrations of H2O2 (10–30 µM) increased protein synthesis without affecting survival. Higher concentrations of H2O2 (100–200 µM) increased apoptosis (assessed by TUNEL). Still higher concentrations of H2O2 (300–1000 µM) caused both apoptosis and necrosis. A hypertrophic concentration of H2O2 (10 µM) increased the activity of ERK1/2, but not that of JNK, p38 kinase or Akt. An apoptotic concentration of H2O2 (100 µM) activated JNK, p38 kinase and Akt, and further activated ERK1/2. The MEK1/2 inhibitor U0126 prevented the hypertrophic effect of 10 µM H2O2. The apoptotic effect of 100 µM H2O2 was inhibited bya dominant-negative JNK adenovirus, and was potentiated by U0126 or an Akt inhibitor. Thus, the concentration-dependent effects of ROS on myocyte hypertrophy and growth are due, at least in part, to the differential activation of specific kinase signaling pathways that regulate hypertrophy and apoptosis. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: H2O2; Apoptosis; Hypertrophy; Cardiac myocyte; Reactive oxygen species; ERK1/2; JNK; c-Jun; p38 kinase; Akt
1. Introduction Reactive oxygen species (ROS) have been implicated in the pathophysiology of myocardial failure [1–3]. Recent investigations using cardiac myocyte systems in culture show that ROS and oxidative stress can cause multiple changes in cell structure and function that are associated with the failing heart, and which appear to be related to the quantity and type of ROS [4]. For example, in neonatal rat cardiac myocytes (NRVM) a small increase in ROS due to inhibition of superoxide dismutase (SOD) causes hypertrophy, whereas a larger increase due to more complete inhibition of SOD causes apoptosis [5]. Similarly, direct addition of ROS leads to apoptosis in NRVM [6,7], H9c2 cells [8,9], and adult rat ventricular myocytes (ARVM) [10], while hypertrophy has been reported in NRVM and H9c2 cells that survived ROS exposure [9]. Other studies using antioxidants * Corresponding author. Tel.: +1-617-638-8706; fax: +1-617-638-8712. E-mail address: [email protected] (W.S. Colucci). © 2003 Elsevier Science Ltd. All rights reserved. DOI: 10.1016/S0022-2828(03)00084-1
suggest that ROS are involved in mediating the hypertrophic and apoptotic effects of pathophysiologically-relevant stimuli such as mechanical strain [11,12], neurohormonal stimulation [13–15] and inflammatory cytokines [14]. While the source(s) of ROS activated by these pathophysiological stimuli is not yet known, several studies have investigated the mechanism by which ROS activate hypertrophic and apoptotic phenotypes in myocytes. ROS are known to activate ERK1/2, p38 kinase, JNK and Akt in many cell types including cardiac myocytes [16]. The role of these pathways in myocyte hypertrophy and apoptosis remains controversial and incompletely defined. However, collectively several studies support a model in which distinct signaling pathways have differential sensitivity to oxidative stress, and thereby lead to the specific myocyte phenotypes of hypertrophy and apoptosis. For example, activation of ERK1/2 in the absence of JNK, p38 kinase or Akt activation occurs in ARVM in an ROS-dependent manner after a-adrenergic receptor stimulation, leading to myocyte hypertrophy in the absence of apoptosis [17]. Similarly, low-level mechanical strain acti-
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vates ERK1/2, but not JNK, and results in hypertrophy in the absence of apoptosis, also in an ROS-dependent manner [12]. In contrast, in ARVM high concentrations of H2O2 cause apoptosis and JNK activation, and overexpression of a dominant-negative SEK1 construct inhibits both JNK activation and apoptosis [10]. H2O2 activates ERK1/2 and JNK in a concentration-dependent manner in ARVM [18]. Taken together, these studies led to our hypothesis that differential, concentration-dependent activation of kinase cascades determines whether oxidative stress leads to myocyte hypertrophy or apoptotic cell death. Accordingly, the first goal of this study was to determine if H2O2 causes distinct, concentration-dependent phenotypes in ARVM. Finding that low and high concentrations of H2O2 cause hypertrophy and apoptosis, respectively, we next tested whether the concentration-dependent effects of ROS on myocyte phenotype are due to differential activation of distinct kinase signaling pathways. 2. Methods 2.1. Isolation of adult rat cardiac myocytes As previously described [19] ARVM (90–95% purity) were isolated from the hearts of adult (200–220 g) male Sprague-Dawley rats, plated at a non-confluent density of 30–50 cells/mm2 on 100-mm plastic culture dishes (Fisher) or 40 × 22-mm2 glass coverslips (Fisher) precoated with laminin (1 µg/cm2, Becton-Dickinson), and maintained in ACCT medium (DMEM; BSA, 2 mg/ml; L-carnitine, 2 mmol/l; creatinine, 5 mmol/l; taurine, 5 mmol/l; penicillin, 100 IU/ml; streptomycin, 10 µg/ml) for 16 h before drug treatment.
H2O2, ARVM were infected with the dn-JNK or b-galactosidase adenovirus at a MOI of 10. 2.4. Cell viability Cells cultured on six well plates were exposed to H2O2 for 24 h. Methylthiazolyldiphenyl-tetrazolium bromide (MTT; 0.2 mg/ml; Sigma) was added and cells were incubated for 3 h at 37 °C in the dark. DMSO and Sorensen’s glycine buffer (0.1 mol/l glycine, 0.1 mol/l NaCl, pH 10.5 with 0.1 N NaOH) were added, and absorbance (OD) was measured at a wavelength of 570 nm. Cell viability was calculated as the percentage of control OD. 2.5. Total myocyte protein ARVM were washed with a phosphate-buffered solution and harvested in a buffer containing 20 mM Tris-HCl (Sigma), 0.5 mM EDTA (Sigma), 0.3 mM EGTA (Sigma), 1 mM dithiothreitol (Sigma) and 1 µM leupeptin (Sigma). Cells were sonicated, precipitated with 10% TCA and resuspended in 0.4 mol/l NaOH buffer. Aliquots were removed for determination of total cellular protein by the Bradford method using Coomassie Blue (BioRad Laboratories). Cellular protein content was normalized to DNA content, which was measured by fluorometric assay after cell lysis. 2.6. 3H-leucine incorporation Incorporation of 3H-leucine was measured over the final 6 h of H2O2 treatment. The activity of 3H-leucine was determined by scintillation counting of samples harvested and precipitated as noted above. 2.7. Histochemistry
2.2. Drug treatments Myocytes were treated with H2O2 (10–1000 µM, Sigma) for 48 h for hypertrophy and 24 h for apoptosis assays, and for 15–60 min for kinase assays. Inhibitors of MEK1/2 (U0126, 10 µM, Calbiochem), p38 kinase (SB20358, 5 µM, Calbiochem) and Akt (1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, 10 µM, Calbiochem) were added 30 min prior to H2O2. 2.3. Adenoviral constructs The T183A, Y185F JNK1 mutant (dn-JNK) [20] (a kind gift from R. Davis, Worcester, MA) was inserted into pAxCAWt cosmid using the Cos-TPC method. pAxCAWt-dnJNK and a DNA-TPC fragment were transfected into HEK 293 cells using the calcium phosphate method and adenoviral plaques were selected. The plaques were amplified in HEK 293 cells and purified using a double cesium chloride gradient. Using the TCID50 method a viral titer was determined. Similar methods were used for the creation of the b-galactosidase vector. Forty-eight hours prior to addition of
TUNEL staining was performed on cells plated on glass coverslips using a Boehringer Mannheim in situ death detection kit according to the manufacturer’s instructions as previously described [19]. The percentage of TUNEL-positive myocytes (relative to total myocytes) was determined by counting 200 cells per coverslip per condition in randomly chosen fields. A total of 1200 cells from six coverslips were counted for each experimental condition. Annexin V (AnV) with propidium iodide (PI) co-staining was performed using a Molecular Probes Vybrant Apoptosis assay kit according to the manufacturer’s instructions. 2.8. Western blotting Cells were collected in lysis buffer (in mM: 1% Triton X100, 0.5% NP-40, 10 Tris, 1 EDTA, 1 EGTA, 150 NaCl, 0.4 PMSF, 0.2 sodium orthovanadate, 1 µg/µl leupeptin). Protein concentration was determined using Bradford method (BioRad). Equal amounts of total protein were separated by SDS-PAGE on 10% gels, and transferred to Immobilin-P transfer membrane (Amersham), which was probed with
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anti-phospho-ERK1/2 (1:1000, Cell Signaling), antiphospho-JNK (1:1000, Santa Cruz), anti-phospho-c-Jun (1:1000, Cell Signaling), anti-phospho-p38 kinase (1:1000, Cell Signaling) or anti-phospho-Akt (1:1000, Cell Signaling) antibodies. Chemiluminescence was quantified by densitometry (Molecular Analyst, Biorad). 2.9. Statistics All data are presented as mean ± S.E.M. Differences among conditions were determined by ANOVA followed by a paired t-test with the Bonferroni correction for multiple comparisons. P values <0.05 were considered significant.
A
3. Results 3.1. Low H2O2 concentrations increase protein synthesis Total myocyte protein and 3H-leucine incorporation were assessed as measures of myocyte hypertrophy. H2O2 at concentrations of 10 or 30 µM increased total protein content by approximately 21–24% (Fig. 1A). At H2O2 concentrations of 100 µM and higher there was a decrease in cell viability associated with cell loss (see below). Likewise, 3H-leucine incorporation was increased by 33–43% at H2O2 concentrations of 10–30 µM (Fig. 1B). 3.2. Cell viability ARVM were exposed to increasing concentrations of H2O2 ranging from 10 to 1000 µM for 24 h and viability was assessed by MTT uptake. There was no decrease in cell viability at H2O2 concentrations of 10 and 30 µM. Cell viability decreased slightly at H2O2 concentrations of 100 µM, and markedly at 300 or 1000 µM (Fig. 2A).
B Fig. 1. Effect of H2O2 on total protein and 3H-leucine incorporation. Myocytes were exposed to H2O2 for 48 h. H2O2 concentrations of 10 and 30 µM increased both total protein (Panel A) and 3H-leucine incorporation (Panel B). At concentrations of 100 µM and higher total protein and 3H-leucine incorporation decreased due to cell loss (see Fig. 2). The data are normalized to DNA content. Shown are the mean from eight to nine experiments. In this and subsequent figures, differences among multiple comparisons were tested by ANOVA followed by paired t-testing with the Bonferroni correction for multiple comparisons (* P < 0.05 vs. control;† P < 0.01 vs. control).
3.3. Higher concentrations of H2O2 stimulate apoptosis H2O2 concentrations of 10–30 µM had no effect on the frequency of apoptotic myocytes as assessed by TUNEL staining. At 100 µM, the frequency of apoptosis increased 4.5-fold (Fig. 2B). AnV (Panel 2C) and PI (Panel 2D) costaining in unfixed cells showed that at an H2O2 concentration of 100 µM AnV staining was increased in the absence of nuclear PI staining, consistent with apoptosis. At an H2O2 concentration of 300 µM there was a marked increase in PI staining, indicating that some of the increase in TUNEL staining at 300 µM H2O2 is due to necrosis. Thus, H2O2stimulated myocyte apoptosis occurs over a relatively narrow concentration range between 30 and 300 µM.
An H2O2 concentration of 10 µM increased the phosphorylation of ERK1/2 2.3-fold with the peak effect at 15 min, but had no consistent effect on p38 kinase or Akt phosphorylation at any time from 5 min to 24 h. There was no increase in c-Jun phosphorylation. In contrast, an H2O2 concentration of 100 µM increased the phosphorylation of p38 kinase, Akt, JNK and c-Jun, and further increased ERK1/2 phosphorylation.
3.5. ERK1/2 mediates hypertrophy at low concentrations of H2O2
3.4. H2O2 concentration-dependent kinase activation H2O2 caused concentration-dependent activation of ERK1/2, JNK, p38 kinase and Akt as assessed by western blotting using antibodies to phosphorylated kinases (Fig. 3).
The MEK1/2 inhibitor U0126 (10 µM) prevented H2O2stimulated (10 µM) hypertrophy (Fig. 4). U0126 prevented H2O2-stimulated activation of ERK1/2, but not the activation of JNK or p38 kinase (data not shown).
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A B
C
D
Fig. 2. Concentration-dependent effect of H2O2 on viability and apoptosis in ARVM. Myocytes were exposed to various concentrations of H2O2 for 24 h and viability was assessed by MTT uptake (Panel A). The data are the mean from three experiments. Myocytes were exposed to H2O2 in concentrations ranging from 10 to 1000 µM and apoptosis was assessed by staining for TUNEL (Panel B), AnV (Panel C) or PI (Panel D). TUNEL staining was performed after exposure for 24 h, and AnV and PI staining were performed after exposure for 6 h. Under control conditions 4.6 ± 1% of myocytes were TUNEL-positive, 3.14 ± 2.3% were AnV-positive/PI-negative and 6.5 ± 1.8% were AnV-positive/PI-positive. Exposure to low concentrations of H2O2 (10–30 µM) had no effect on TUNEL, AnV or PI staining. An H2O2 concentration of 100 µM increased the frequencies of TUNEL and AnV-positive/PI-negative staining by 4.5- and 8.8-fold, respectively, without affecting the rate of AnV-positive/PI-positive staining. An H2O2 concentration of 300 µM significantly increased AnV-positive/PI-positive staining consistent with necrosis or other form of cell death. The data are the mean from six (TUNEL) or three (AnV and PI) experiments (* P < 0.05 vs. control; † P < 0.01 vs. control).
3.6. JNK promotes apoptosis at high concentrations of H2O2, whereas Akt and ERK1/2 are anti-apoptotic Apoptosis stimulated by a high concentration of H2O2 (100 µM) was decreased approximately 57% by infection with an adenoviral vector for a dominant-negative JNK (Fig. 5A). In contrast, the MEK1/2 inhibitor U0126 (Fig. 5B) and the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Fig. 5C) each increased H2O2-stimulated apoptosis by approximately 60%. The p38 kinase inhibitor SB20358 had no consistent effect on H2O2-stimulated apoptosis (Fig. 5D). 4. Discussion There are four major new findings of this study. First, H2O2 caused concentration-dependent effects on ARVM phenotype, resulting in hypertrophy at low concentrations
and apoptosis at higher concentrations. Second, these effects on phenotype were associated with concentration-dependent activation of four kinase signaling pathways—ERK1/2, JNK, p38 kinase and Akt. Third, H2O2-stimulated hypertrophy was mediated by ERK1/2, whereas H2O2-stimulated apoptosis was mediated by JNK. Finally, ROS-dependent activation of ERK1/2 and Akt exerted anti-apoptotic actions, which opposed the pro-apoptotic effect of JNK. The concentration-dependent hypertrophic and apoptotic phenotypes induced by H2O2 in ARVM is similar to what we have previously reported in NRVM in response to SOD inhibition or mechanical strain. In neonatal rat cardiac myocytes we found that an increase in ROS due to inhibition of SOD or addition of xanthine oxidase resulted in hypertrophy and apoptosis: hypertrophy occurred at low levels of oxidative stress, whereas apoptosis occurred at higher levels of oxidative stress [5,12]. Likewise, ROS have also been implicated in mediating both hypertrophy and apoptosis in adult
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Fig. 3. Concentration-dependent effects of H2O2 on kinase activity. Myocytes were exposed to H2O2 concentrations of 10 or 100 µM for 15 min, and the phosphorylation of ERK1/2, JNK, p38 kinase and Akt was determined by western blotting. The phosphorylation of c-Jun was determined at 60 min. H2O2 at a concentration of 10 µM, increased the activation of ERK1/2 (Panel A), but not JNK (Panel B), c-Jun (Panel C), p38 kinase (Panel D) or Akt (Panel E). At an H2O2 concentration of 100 µM, there was activation of p38 kinase, JNK, c-Jun and Akt, and more intense activation of ERK1/2 (* P < 0.05 vs. control; † P < 0.01 vs. control).
effects of a-adrenergic receptor agonists and endothelin in ARVM. The magnitude of the hypertrophic effect of 10–30 µM H2O2 was less than that of serum, but similar to what we have previously observed for a-adrenergic stimulation in ARVM [13]. Aoki et al. [10] showed that ROS can cause apoptosis in ARVM. In the present study we found that H2O2 can cause both hypertrophy and apoptosis in a concentrationdependent manner in ARVM. Taken together with the prior studies in NRVM, we conclude that the effects of ROS on myocyte phenotype are determined, at least in part, by the concentration of ROS.
Fig. 4. Prevention of H2O2-stimulated hypertrophy by the MEK1/2 inhibitor U0126. Myocytes pretreated with U0126 (10 µM; 30 min) were exposed to a hypertrophic concentration of H2O2 (10 µM) for 24 h. Data depict the mean total cellular protein content from nine experiments (* P < 0.05 vs. control; † P < 0.05 vs. H2O2).
cardiac myocytes. Amin et al. [13] and Tanaka et al. [15] have shown that ROS are involved in mediating the hypertrophic
Although high concentrations of ROS can cause toxic cell injury, there is now evidence that lower, sub-toxic concentrations of ROS can act as intracellular signaling molecules [4,21]. ROS activate a variety of kinase signaling pathways that have been implicated in the regulation of myocyte hypertrophy and/or apoptosis, including ERK1/2, JNK, p38 kinase and Akt. We found that the pattern of kinases activated was related to the concentration of H2O2, a finding that is generally similar to that of Wei et al. [18]. At low concentrations,
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B
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D Fig. 5. Effects of kinase inhibitors on H2O2-stimulated apoptosis. Panel A. Myocytes were infected with an adenoviral vector for a dominant-negative mutant of JNK1 for 24 h prior to exposure to H2O2 for an additional 24 h. Apoptosis was assessed by TUNEL staining. Data are the mean from three experiments (* P < 0.01 vs. control; † P < 0.05 vs. H2O2). Panel B. Pretreatment with the MEK1/2 inhibitor U0126 (10 µM; 30 min). Data are the mean from four experiments (* P < 0.01 vs. control; † P < 0.01 vs. H2O2). Panel C. Pretreatment with the Akt inhibitor 1L-6-hydroxymethylchiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (10 µM; 30 min). Data are the mean from four experiments (* P < 0.01 vs. control; † P < 0.01 vs. H2O2). Panel D. Pretreatment with the p38 kinase inhibitor SB20358 (5 µM; 30 min). Data are the mean from five experiments (* P = 0.0083 vs. control).
H2O2 activated primarily ERK1/2, but not JNK, p38 kinase or Akt. With higher concentrations, we found increased activation of ERK1/2, along with activation of JNK, p38 kinase and Akt, and increased c-Jun phosphorylation. The hypertrophic effect of 10 µM H2O2 was markedly decreased by the MEK1/2, but not the JNK inhibitor. We found that the Akt inhibitor also attenuated hypertrophy. However, given the lack of consistent activation of Akt by 10 µM H2O2, it is possible that this reflects non-selective effect of the Akt inhibitor on ERK1/2 signaling. Therefore, although the data support an important role for ERK1/2 in mediating the hypertrophic effect of 10 µM H2O2, we can neither implicate nor exclude a role for Akt with these data. The apoptotic effect of 100 µM H2O2 was inhibited by infection with an adenovirus carrying dominant-negative JNK. These findings are consistent with the demonstration by Aoki et al. [10] that H2O2-stimulated apoptosis in ARVM is prevented by overexpression of a dominant-negative SEK1. Likewise, JNK has been implicated in mediating ROSstimulated apoptosis in the H9c2 muscle cell line [8,22]. However, there is also evidence that JNK can mediate antiapoptotic effects in neonatal rat cardiac myocytes [23,24] and embryonic stem cell-derived cardiac myocytes [25]. These divergent results may be due to differences in kinase signaling pathways related to developmental stage or culture conditions. We did not assess hypertrophy at higher concentrations of H2O2 that caused apoptosis. However, Chen et al. [9] have suggested that apoptotic concentrations of H2O2 may also be associated with hypertrophy of the surviving cells. Like other investigators, we found that ERK1/2 or Akt pathways activated at high concentrations of H2O2 play a protective role against H2O2-induced apoptosis [6,26]. These findings suggest that the regulation of apoptosis by ROS involves multiple kinase signaling pathways, with the net effect reflecting a balance between the pro-apoptotic effect of JNK and the anti-apoptotic actions of ERK1/2 and Akt. Taken together, these data suggest that the level of ROS is an important determinant of myocyte phenotype via the differential activation and interplay of multiple kinase signaling pathways. There is increasing evidence that a variety of stimuli including mechanical strain [11,12], neurohormonal stimulation [13–15] and inflammatory cytokines [14] can increase the level of ROS in cardiac myocytes. The present findings help to elucidate the complex role that ROS play in mediating myocyte phenotype and provide a mechanism by which a stimulus may exert diverse effects on myocyte hypertrophy and apoptosis. Acknowledgements We are grateful to Paul O. Kwon for his technical expertise and helpful discussions. We also thank Jing Wang and Xinxin Guo for their excellent technical assistance. This work was supported by NIH grants HL61639 and HL20612 (W.S.C.), HL03878 (D.B.S.), an NIH Cardiovascular Scientist Training Grant HL07224 (S.H.K. and D.R.P.); and a grant from the Swiss National Science Foundation (A.R.).
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