Evidence of Altered Guinea Pig Ventricular Cardiomyocyte Protein Expression and Growth in Response to a 5 min in vitro Exposure to H2O2 Vidya Seenarain,† Helena M. Viola,† Gianina Ravenscroft,‡ Tammy M. Casey,§ Richard J. Lipscombe,§ Evan Ingley,‡ Nigel G. Laing,‡ Scott D. Bringans,§ and Livia C. Hool*,† School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, 6009 Australia, Proteomics International, Lotterywest State Biomedical Facility, Perth, Western Australia, 6000 Australia, and The Centre for Medical Research and The Western Australian Institute for Medical Research, Perth, Western Australia, 6000 Australia Received December 11, 2009
Oxidative stress and alterations in cellular calcium homeostasis are associated with the development of cardiac hypertrophy. However, the early cellular mechanisms for the development of hypertrophy are not well understood. Guinea pig ventricular myocytes were exposed to 30 µM H2O2 for 5 min followed by 10 units/mL catalase to degrade the H2O2, and effects on protein expression were examined 48 h later. Transient exposure to H2O2 increased the level of protein synthesis more than 2-fold, assessed as incorporation of [3H]leucine (n ) 12; p < 0.05). Cell size was increased slightly, but there was no evidence of major cytoskeletal disorganization assessed using fluorescence microscopy. Changes in the expression of individual proteins were assessed using iTRAQ protein labeling followed by mass spectrometry analysis (LC-MALDI-MSMS); 669 proteins were identified, and transient exposure of myocytes to H2O2 altered expression of 35 proteins that were predominantly mitochondrial in origin, including TCA cycle enzymes and oxidative phosphorylation proteins. Consistent with changes in the expression of mitochondrial proteins, transient exposure of myocytes to H2O2 increased the magnitude of the mitochondrial NADH signal 10.5 ( 2.3% compared to cells exposed to 0 µM H2O2 for 5 min followed by 10 units/mL catalase (n ) 8; p < 0.05). In addition, metabolic activity was significantly increased in the myocytes 48 h after transient exposure to H2O2, assessed as formation of formazan from tetrazolium salt. We conclude that a 5 min exposure of ventricular myocytes to 30 µM H2O2 is sufficient to significantly alter protein expression, consistent with the development of hypertrophy in the myocytes. Changes in mitochondrial protein expression and function appear to be early sequelae in the development of hypertrophy. Keywords: Oxidative stress • cardiac hypertrophy • mitochondria • protein expression • NADH • metabolic activity
Introduction Oxidative stress is a feature of cardiovascular disease. Excessive reactive oxygen species (ROS) generation can lead to irreversible cell damage or death.1 However, it is now well recognized that ROS can also act as signaling molecules able to stimulate and modulate a variety of biochemical and genetic systems, including the regulation of signal transduction pathways, gene expression, and proliferation.1 One of these, hydrogen peroxide (H2O2), is believed to interact with cell signaling pathways by way of modification of key thiol groups * To whom correspondence should be addressed: Physiology M311, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009 Australia. Telephone: 61 8 6488 3307. Fax: 61 8 6488 1025. E-mail:
[email protected]. † The University of Western Australia. ‡ The Centre for Medical Research and The Western Australian Institute for Medical Research. § Proteomics International. 10.1021/pr9011393
2010 American Chemical Society
on proteins that possess regulatory functions.2 H2O2 is a stable, lipid soluble molecule that can easily cross membranes. Elevations in cellular H2O2 and superoxide levels contribute to the development and progression of ischemic heart disease, cardiac hypertrophy, and congestive heart failure through activation of hypertrophic signaling pathways.3-5 A number of signaling pathways involved in hypertrophic growth are also activated by an increase in the intracellular calcium concentration. Phenotypic hypertrophic remodeling occurs because alterations to calcium transients result in activation of calcium-dependent pathways, including the NFAT and CaMK pathways.6-9 A small increase in the intracellular calcium concentration (insufficient to cause further release of calcium from intracellular stores) is sufficient to cause translocation of NFAT from the cytoplasm to the nucleus to initiate the transcription of proteins.10,11 Transgenic mice expressing Journal of Proteome Research 2010, 9, 1985–1994 1985 Published on Web 02/05/2010
research articles constitutively active forms of calcineurin or NFAT develop cardiac hypertrophy, resulting in heart failure and sudden death.12 The mitochondria are a significant source of ROS in the heart. Persistent production of superoxide and H2O2 by the mitochondria has been associated with the pathogenesis of ischemic heart disease and development of cardiac hypertrophy.13 The mechanisms for the increase in cellular ROS levels and development of cardiac hypertrophy continue to be poorly understood. A persistent oxidative state can arise in cardiac myocytes when ROS released from mitochondria then trigger further production of ROS from nearby mitochondria, a phenomenon termed “ROS-induced ROS release”.14-16 We have demonstrated previously that a 5 min exposure of ventricular myocytes to 30 µM H2O2 followed by 10 units/mL catalase to degrade the H2O2 is sufficient to induce further release of superoxide from mitochondria without causing apoptosis or necrosis.16 The increase in the level of superoxide occurs because of an increase in the diastolic calcium level as a result of direct activation of the L-type Ca2+ channel (ICa-L) by H2O2. ICa-L current density was found to be persistently increased for at least 8 h after the 5 min exposure to H2O2. The channel remains in an oxidized state due to an increased rate of uptake of calcium by the mitochondria and persistent production of superoxide by the mitochondria.16 We have proposed that this may be a mechanism for induction of hypertrophy associated with oxidative stress. The purpose of this study was to determine whether a transient exposure of ventricular myocytes to H2O2 is sufficient to alter protein expression and function consistent with the development of cardiac hypertrophy. The guinea pig was the animal model of choice because ion channel protein expression (that determines intracellular calcium homeostasis) is similar to that in human heart.17 In addition, our previous work demonstrating alterations in ICa-L current density, intracellular calcium homeostasis, and mitochondrial superoxide production in response to transient exposure to H2O2 was performed in guinea pig ventricular myocytes.16 We found that exposure of ventricular myocytes to 30 µM H2O2 for 5 min followed by 10 units/mL catalase significantly increased the level of protein synthesis (assessed as incorporation of [3H]leucine) in the myocytes 48 h later. We used iTRAQ-facilitated labeling in which amine-specific, stable isotope reagents label all peptides in the samples, enabling simultaneous identification and quantitation. This is achieved through relative comparison of the reporter ions that are produced upon fragmentation of the peptides. This study identified alterations in expression of 35 proteins that were predominantly mitochondrial tricarboxylic acid (TCA) cycle enzymes and oxidative phosphorylation proteins. The results provide significant insight into the early mechanisms associated with the development of cardiac hypertrophy involving increased oxidative stress.
Experimental Procedures Cell Isolation. Ventricular myocytes were isolated from 6-8week-old adult Tricolour guinea pigs of either sex using a modification of the collagenase dissociation method.18 Guinea pigs were anesthetized with intraperitoneal injection of pentobarbitone sodium (240 mg/kg) prior to excision of the heart, as approved by The Animal Ethics Committee of The University of Western Australia in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NH&MRC, 7th ed., 2004). 1986
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Measurement of Incorporation of [ H]Leucine. Freshly isolated myocytes were added to flasks with DMEM supplemented with 1 mL/100 mL penicillin/streptomycin and incubated in a 5% CO2 incubator at 37 °C. Following incubation for 1 h, myocytes were exposed to either 0 µM H2O2 for 5 min and then 10 units/mL catalase, 30 µM H2O2 for 5 min and then 10 units/mL catalase, 2 µM nisoldipine and then 30 µM H2O2 for 5 min and then 10 units/mL catalase, 20 µM dantrolene and then 30 µM H2O2 for 5 min and then 10 units/mL catalase, 10 µM KN-62 and then 0 µM H2O2 for 5 min and then 10 units/ mL catalase, 10 µM KN-62 and then 30 µM H2O2 for 5 min and then 10 units/mL catalase, or 100 µM phenylephrine. Following overnight incubation, the culture medium was changed to fresh DMEM supplemented with 0.1% gentamycin, 10% FCS, and 1 µCi/mL [3H]leucine (GE Healthcare) and the mixture incubated at 37 °C for a further 24 h. Culture medium was then removed, and cells were washed twice with HBS containing 5.33 mM KCl, 0.41 mM MgSO4, 139 mM NaCl, 5.63 mM Na2HPO4, 5 mM glucose, 20 mM HEPES, 2 mM glutamine, 2.5 mM Ca(NO3)2, and 1 mL/100 mL penicillin/streptomycin (pH adjusted to 7.4 with NaOH). Myocytes were then incubated with 5% trichloroacetic acid for 30 min at 4 °C to precipitate proteins. Precipitates were washed twice with cold ddi H2O, resuspended in 0.4 M NaOH, and incubated at 4 °C for 1 h. Radioactivity was measured in a liquid scintillation counter (Wallac 1409 instrument). Measurement of Cell Area. ImageJ (Microsoft Java 1.42, National Institutes of Health, Bethesda, MD) was used to quantify myocyte size 48 h following treatment. Freshly isolated myocytes were allowed to become adherent to polylysinecoated 96-well plates for approximately 1 h before being treated with either 0 µM H2O2 for 5 min and then 10 units/mL catalase (n ) 127) or 30 µM H2O2 for 5 min and then 10 units/mL catalase (n ) 129). Forty-eight hours following treatments, myocytes were fixed in 2% paraformaldehyde. Images of the myocytes were captured using confocal microscopy. ImageJ was used to measure the cell size of each image by manually tracing myocytes. Myocyte area was initially recorded by ImageJ in pixels. Myocyte area was then converted from pixels to square micrometers using a scale of 36 pixels ) 1 µm2. Size measurements for all myocytes were taken blindly. Fluorescence Microscopy. Forty-eight hours post-treatment with either 0 µM H2O2 for 5 min and then 10 units/mL catalase or 30 µM H2O2 for 5 min and then 10 units/mL catalase, myocytes were fixed with 2% paraformaldehyde, washed, permeabilized with 0.5% saponin, and blocked with a 10% FCS/ 1% BSA mixture for 15 min. Myocytes were incubated for 60 min at room temperature (RT) with phalloidin-TRITC to label F-actin or desmin antibody (D033, Dako) conjugated with the Zenon AlexaFluor mouse IgG1 594 labeling kit (Invitrogen). Samples were then washed and incubated for 5 min with 4′,6diamidino-2-phenylindole (DAPI) to label the nuclei, mounted with Hydromount, and imaged on a Bio-Rad (Hercules, CA) confocal microscope (model MRC1000/1024). iTRAQ Labeling of Proteins. Freshly isolated myocytes were plated in suspension in flasks with DMEM supplemented with 1 mL/100 mL penicillin/streptomycin and incubated in a 5% CO2 incubator at 37 °C. After being incubated for 1 h, myocytes were exposed to either 0 µM H2O2 for 5 min and then 10 units/ mL catalase or 30 µM H2O2 for 5 min and then 10 units/mL catalase at 37 °C. Myocytes were cultured overnight, and then the culture medium was changed to fresh DMEM supplemented with 0.1% gentamycin and 10% FCS. Myocytes were
Altered Ventricular Cardiomyocyte Protein Expression and Growth then cultured for a further 24 h in a 5% CO2 incubator at 37 °C. Following culture overnight, culture medium was removed and cells were washed twice with HBS. Lysis buffer containing 50 mM Tris (pH 7), 0.5 mM EDTA, 20% glycerol, and a protease inhibitor cocktail tablet (EDTA-free) was added to cells before each sample was sonicated on ice twice for 10 s with a 5 s delay between bursts. Samples were centrifuged at 13000g for 10 min at 4 °C. Supernatants were removed and used for iTRAQ-facilitated proteomics. Two hundred micrograms of protein from cells treated with 30 µM H2O2 for 5 min and then 10 units/mL catalase and 200 µg of protein from cells treated with 0 µM H2O2 for 5 min and then 10 units/mL catalase were subjected to acetone precipitation and incubated for 1 h at -20 °C. The precipitated protein was resuspended in 0.5 M triethylammonium bicarbonate (pH 8.5), before reduction and alkylation according to the iTRAQ protocol (Applied Biosystems, Foster City, CA). Samples were centrifuged at 13000g for 10 min at room temperature before the supernatant was assayed for protein concentration (BioRad protein assay kit). Fifty-eight micrograms of each sample was digested with trypsin overnight and labeled according to the manufacturer’s instructions. Peptides from samples that had been treated with 30 µM H2O2 for 5 min and then 10 units/ mL catalase were labeled with iTRAQ reagent 117, and peptides from samples that had been treated with 0 µM H2O2 for 5 min and then 10 units/mL catalase were labeled with iTRAQ reagent 115. The iTRAQ derivatization reaction was quenched by the addition of 1 mL of water before samples were combined, desalted on a Strata-X 33 µm polymeric reverse phase column (Phenomenex, Torrance, CA), and dried in a vacuum concentrator. Strong Cation Exchange Chromatography. Peptides were separated by strong cation exchange chromatography on an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA) using a PolySulfethyl column (4.6 mm × 100 mm, 5 µm, 300 Å, Nest Group, Southborough, MA). Peptides were eluted with a linear gradient of buffer B [1 M KCl, 10% acetonitrile, and 10 mM KH2PO4 (pH 3)]. A total of 40 fractions were collected and pooled into eight fractions, desalted, and dried. Reverse Phase Nano LC-MALDI-MS/MS. Peptide fractions were separated on a C18 PepMap100, 3 µm column (LC Packings, Sunnyvale, CA) with a gradient of acetonitrile in 0.1% trifluoroacetic acid using the Ultimate 3000 nano HPLC system (LC Packings-Dionex, Sunnyvale, CA). The eluent was mixed with matrix solution (5 mg/mL R-cyano-4-hydroxycinnamic acid) and spotted onto a 384-well Opti-TOF plate (Applied Biosystems, Framingham, MA) using a Probot Micro Fraction Collector (LC Packings, San Francisco, CA). Peptides were analyzed on a 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA) operated in reflector positive mode. MS data were acquired over a mass range of m/z 800-4000, and for each spectrum, a total of 400 shots were collected. A job-wide interpretation method selected the 20 most intense precursor ions above a signal-to-noise ratio of 20 from each spectrum for MS/MS acquisition but only in the spot where their intensity was at its peak. MS/MS spectra were acquired with 4000 laser shots per selected ion with a mass range of 60 to the precursor ion at -20 amu. Data Analysis. Protein identification and quantification were performed using ProteinPilot version 2.0.1 (Applied Biosytems, Foster City, CA). MS/MS spectra were searched against a Cavia porcellus database assembly downloaded from http://www. ensembl.org (release 56, 19774 entries) and formatted for use with ProteinPilot. To estimate the false discovery rate (FDR), a
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decoy database search against a concatenated database containing forward and randomized entries of the C. porcellus database was performed. The FDR was calculated as follows: [number of random hits/(total number of hits - number of random hits)] × 100.19 Identified proteins were grouped by the software to minimize redundancy. All peptides used for the calculation of protein ratios were unique to the given protein or proteins within the group; peptides that were common to other isoforms or proteins of the same family that were reported separately were ignored. Search parameters were as follows: sample type, iTRAQ 4plex (peptide labeled); Cys alkylation, MMTS; Digestion, trypsin; Instrument, 4800; Special factors, none; Species, none; Quantitate tab, checked; ID focus, Biological modifications; Search effort, thorough; Detected protein threshold (unused ProtScore), 1.3 (which corresponds to proteins identified with >95% confidence). Proteins identified only by a single unique sequence were removed. For each reported protein ratio, the program calculated a p value to assess whether changes in protein expression were real. The p value reports the probability that the null hypothesis of whether “the observed value is different from unity by chance” is true. A p value of e0.05 indicates statistically significant differential expression. The p values represent the variation in the reported iTRAQ ratios for all the peptides of the associated protein and do not relate to either biological variation (across animals) or technical reproducibility. They are the standard measure used by ProteinPilot to determine significance. Measurement of the Mitochondrial NADH Level. Autofluorescence of reduced nicotinamide adenine dinucleotide (NADH) was monitored in intact guinea pig ventricular myocytes in HBS at 37 °C as described previously.20,21 Fluorescence with excitation at 365 nm and emission at 460 and 535 nm was measured on a Hamamatsu Orca ER digital camera attached to an inverted Nikon TE2000-U microscope. Metamorph 6.3 was used to quantify the signal by manually tracing myocytes. Ratiometric 460 nm/535 nm fluorescence was plotted relative to the pretreatment fluorescence assigned a value of 1.0. Fluorescence ratios recorded over 5 min before and immediately following treatments were averaged, and alterations in the fluorescence ratio were reported as percentage increases from the baseline average. To confirm that the NADH signal was indicative of mitochondrial NADH production, 20 µM oligomycin and 4 µM FCCP were added at the end of each experiment. The addition of 20 µM oligomycin and 4 µM FCCP was used to obtain maximum and minimum NADH fluorescence intensity, respectively. Measurement of Metabolic Activity. Reduction of 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) is dependent upon the presence of reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), as well as intact mitochondrial electron transport. The basis for this assay is the cleavage of the yellow tetrazolium salt (MTT) to purple formazan crystals by complex I of the electron transport chain within the mitochondria.22 An increase in absorbance represents an increase in the level of formazan production and therefore an increase in metabolic activity. Myocytes were treated with 0 or 30 µM H2O2 for 5 min followed by 10 units/mL catalase and cultured for 48 h in suspension in 96-well plates.21 MTT was added to each well (final concentration of 0.5 mg/mL). The rate of increase in absorbance was immediately measured using a spectrophotometer (PowerWave XS) at 570 nm with a reference Journal of Proteome Research • Vol. 9, No. 4, 2010 1987
research articles wavelength of 620 nm at 37 °C. The rate of increase in absorbance of treated myocytes was expressed as a percentage of the rate of increase in absorbance of untreated (control) myocytes. Each n represents the number of replicates for each treatment group from three different animals. Addressing Biological and Analytical Reproducibility. All experiments were performed on myocytes isolated from Tricolour guinea pig hearts. For measurement of incorporation of [3H]leucine, myocytes were cultured in flasks. Each n represents the number of culture flasks for each treatment group. Each culture flask contained cells from one animal, and some flasks were replicates of cells from the same animal. Between four and seven animals were used for each treatment group for measurement of incorporation of [3H]leucine. Proteomics studies were performed on myocytes isolated from at least four guinea pigs. Myocytes were treated in four or more culture flasks per treatment group as described above and cultured for 48 h, and protein was extracted and pooled into the treatment groups for iTRAQ analysis. For the MTT assay, cells were cultured in 96-well plates. Each n represents the number of individual wells of myocytes. For the NADH functional assay, studies were performed on individual myocytes. The minimum number of cells required to ensure statistical power of comparison and prevent a Type II error was studied. Experiments were performed on myocytes from at least three animals per treatment. The number of experiments was determined in consultation with a statistician. Results are reported as means ( the standard error of the mean (SEM). Statistical comparisons of responses between unpaired data were made using the Student’s t test or between groups of cells using ANOVA and the Tukey’s post hoc test (GraphPad Prism version 3.02).
Results Transient Exposure of Ventricular Myocytes to H2O2 Increases the Rate of Incorporation of [3H]Leucine. When ventricular myocytes are exposed to increasing concentrations of H2O2 titrating up from 0 µM, a significant increase in the level of cellular superoxide is reproducibly detected at H2O2 concentrations between 20 and 30 µM.16 We exposed ventricular myocytes to either 0 µM H2O2 for 5 min and then 10 units/ mL catalase or 30 µM H2O2 for 5 min and then 10 units/mL catalase and measured the total level of protein expression as incorporation of [3H]leucine into the myocytes 48 h later. Since it has been demonstrated that a significant increase in the influx of calcium through the L-type Ca2+ channel (ICa-L) is responsible for the increase in mitochondrial superoxide, we also exposed cells to ICa-L antagonist nisoldipine. Exposure to H2O2 was associated with a >2-fold increase in the rate of incorporation of [3H]leucine that was significantly attenuated in the presence of nisoldipine (Figure 1). The increase in the rate of incorporation of [3H]leucine was unaffected by the RyR inhibitor dantrolene but could be attenuated with application of the CaMK II inhibitor KN-62. Consistent with previously published work, the hypertrophic agent phenylephrine significantly increased the rate of incorporation of [3H]leucine into the myocytes.23 We examined whether the increase in the rate of incorporation of [3H]leucine was due to a decrease in the rate of degradation of the protein. We exposed myocytes first to fresh DMEM supplemented with 1 µCi/mL [3H]leucine (see Experimental Procedures). The cells were washed of excess [3H]leucine the next morning, then exposed to 0 µM H2O2 for 5 min 1988
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Figure 1. Transient exposure to H2O2 increases the level of protein synthesis in cardiac myocytes. Incorporation of [3H]leucine in cardiac myocytes after exposure to 0 or 30 µM H2O2 for 5 min followed by 10 units/mL catalase (Cat) in the presence and absence of 2 µM nisoldipine (Nisol), 20 µM dantrolene (Dant), or 10 µM KN-62 as indicated. Phenylephrine (PE) was used as a positive control. Incorporation of [3H]leucine into cardiac myocytes prior to exposure to 0 or 30 µM H2O2 for 5 min followed by 10 units/mL catalase is shown in the inset at the right (for further explanation, see the text). Each n represents the number of culture flasks of cells for each treatment group.
and then 10 units/mL catalase or 30 µM H2O2 for 5 min and then 10 units/mL catalase, and incubated for a further 48 h. Exposure to H2O2 did not alter the incorporation of [3H]leucine into the myocytes (Figure 1, inset). These results suggest that a 5 min exposure of ventricular myocytes to 30 µM H2O2 can induce an increase in the level of protein synthesis 48 h later as a result of increased calcium influx through ICa-L and activation of a Ca2+-calmoldulin-dependent signal pathway. Evidence of Cardiomyocyte Hypertrophy after Transient Exposure to H2O2. Features of cardiac hypertrophy include an increase in the total level of protein synthesis, an increase in cell size, and evidence of disorganization in cytoskeletal proteins.7,8 We examined whether a 5 min exposure of myocytes to H2O2 altered cell size 48 h later. We calculated cell size after manual identification and tracing of myocytes using ImageJ (as described in Experimental Procedures). Exposure to 30 µM H2O2 was associated with a small but significant increase in cell size compared to cells exposed to 0 µM H2O2 and catalase (Figure 2A). We also examined whether a transient exposure of myocytes to H2O2 was sufficient to induce alterations in the organization of cytoskeletal proteins. Forty-eight hours after being exposed to either 0 or 30 µM H2O2 for 5 min and then 10 units/mL
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Altered Ventricular Cardiomyocyte Protein Expression and Growth
Figure 2. Transient exposure to H2O2 increases cell size without evidence of disorganization of actin filaments. (A) Means ( SEM of cell size for cells exposed to 0 or 30 µM H2O2 for 5 min followed by 10 units/mL catalase (Cat) as indicated (see Experimental Procedures for further details). (B and C) Cells exposed to 0 or 30 µM H2O2 for 5 min followed by 10 units/mL catalase, fixed, and stained for phalloidin (red, F-actin) and DAPI (blue, nucleus) (B) or desmin (red) and DAPI (blue, nucleus) (C) and viewed by confocal laser microscopy. Table 1. Proteins Identified as Statistically Significantly Up- or Downregulated by Searching against the C. porcellus Database (ensemble release 56, 19774 entries) total no. of proteins identified
no. of upregulated proteins (% of total)
no. of downregulated proteins (% of total)
total no. of proteins differentially expressed (% of total)
669
20 (3.0%)
15 (2.2%)
35 (5.2%)
catalase, cells were fixed on glass coverslips and exposed to phalloidin labeled with TRITC (n ) 7) or desmin antibody (n ) 10). The cells were also stained with DAPI to characterize the nucleus. The cells were viewed using confocal laser microscopy. Treatment with H2O2 was not associated with an observable disorganization of actin filaments compared with cells treated with catalase only (Figures 2B and 2C). Transient Exposure of Ventricular Myocytes to H2O2 Alters Protein Expression. Our results indicate that transient exposure of myocytes to H2O2 increases the total amount of
protein assessed as incorporation of [3H]leucine (Figure 1). We used the iTRAQ labeling technique to identify and quantitate the expression levels of proteins 48 h after myocytes had been treated with either 0 or 30 µM H2O2 for 5 min followed by 10 units/mL catalase. MS/MS spectra were analyzed by searching against a C. porcellus database using ProteinPilot version 2.0.1. A total of 669 proteins were significant, nonredundant matches at the 95% confidence level, and 559 proteins were significant at the 99% confidence level (Unused Prot Score of >2.0) (Table 1 and Table S1 of the Supporting Information). The false Journal of Proteome Research • Vol. 9, No. 4, 2010 1989
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Table 2. Proteins Identified as Upregulated by iTRAQ Analysis protein name
mitochondrial, TCA cycle malate dehydrogenase succinyl-CoA:3-ketoacid-coenzyme A transferase 1 mitochondrial, oxidative phosphorylation NADH dehydrogenase [ubiquinone] flavoprotein 1 ATP synthase subunit ε-like protein, mitochondrial mitochondrial, other functions propionyl-CoA carboxylase β-chain voltage-dependent anion-selective channel protein 2 (VDAC-2) dihydrolipoyl dehydrogenase cytoplasmic, sarcomeric β-myosin heavy chain myosin light chain 3 myosin regulatory light chain 2, ventricular/cardiac muscle isoform creatine kinase M-type troponin C, slow skeletal and cardiac muscles (TN-C) cytoplasmic, glycolysis/gluconeogenesis enolase cytoplasmic, other functions protein disulfide isomerase cytosol aminopeptidase (EC 3.4.11.1) (leucine aminopeptidase) elongation factor 1-R 1 (EF-1-R-1) putative phospholipase B-like 2 precursor nuclear histone H1.4 (histone H1b) zinc finger CCCH domain-containing protein 4 miscellaneous unnamed protein (novel)
% coverage
no. of peptidesa
accession numberb
117:115c
Unused Prot Scored
p valuee
81.1 51.3
56 30
19095 11673
1.1392 1.1023
86.25 46.39
0.0005 0.0128
33.8 54.9
14 4
05984 11149
1.2297 1.1814
17.76 6.27
0.0337 0.0076
29.5 32.0
9 11
10724 02499
1.2229 1.0877
12.17 11.26
0.0037 0.0462
39.3
22
04776
1.1509
32.59
0.0119
57.0 74.25 94.6
140 47 38
06354 11344 09620
1.1855 1.2384 1.1983
215.86 74.25 59.70
0.0002 0.0000 0.0001
64.3 16.8
35 2
16471 02955
1.1084 1.3879
53.59 2.57
0.0219 0.042
51.2
15
05241
1.1559
23.39
0.0292
47.0 18.9 17.9 6.0
28 11 7 3
11016 11358 19648 12800
1.0763 1.1626 1.2774 1.0699
40.35 14.76 8.02 3.50
0.0472 0.0432 0.0248 0.0463
20.8 9.2
4 5
17464 11345
1.3731 1.3524
7.22 1.84
0.0054 0.0237
16.7
3
18235
1.1713
2.06
0.0151
a Number of unique peptide sequences assigned to the protein. b All accession numbers were prefaced by ENSCPOP000000. c 117:115 is the average protein ratio of cells treated with 30 µM H2O2 for 5 min and then 10 units/mL catalase to cells treated with 0 µM H2O2 for 5 min and then 10 units/mL catalase. d The Unused Prot Score is a measure of all the peptide evidence for a protein that is not better explained by a higher-ranking protein. For proteins to be identified with >95%, the required Unused Prot Score is 1.3. e Proteins were only considered to be statistically significantly up- or downregulated if p < 0.05.
discovery rates were estimated to be 11.5 and 4.5%, respectively. A total of 35 proteins were detected with altered expression. Of these, 20 proteins were significantly upregulated (Table 2) while 15 were significantly downregulated (Table 3). The proteins with altered expression were classified according to their subcellular locations being mitochondrial, cytoplasmic, or nuclear proteins. The mitochondrial proteins were further classified according to their functions within the organelle which included TCA cycle enzymes and proteins involved in oxidative phosphorylation. Cytoplasmic proteins were subdivided into glycolytic/gluconeogenic proteins and sarcomeric proteins. Figure 3 demonstrates the cellular localization of all proteins with altered expression after H2O2 exposure. iTRAQfacilitated labeling is semiquantitative and determines significant alterations in expression only. Our results indicate that the greatest impact of the H2O2 exposure was on the mitochondria, with 15 of the 35 proteins with significantly altered expression levels having a mitochondrial origin. In addition, one of the 35 proteins with altered expression was a glycolytic/ gluconeogenic protein. Therefore, 16 of the 35 proteins (46%) affected by the exposure of the cardiac myocyte to H2O2 were proteins directly involved in cellular energy production in the myocytes, including glycolysis, the TCA cycle, and oxidative phosphorylation. Sarcomeric cytoplasmic protein expression was also significantly altered, consistent with the measured increase in cell size (Figure 2A). In addition, although we did not detect 1990
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cytoskeletal disarray, alterations in the expression of β-myosin heavy chain and troponin C are typical features of myofibrillar disarray observed in advanced cardiac hypertrophy.24 Our data may represent early changes in protein expression that then progress to the disorganized cytoskeleton observed in the hypertrophic heart. Transient Exposure of Ventricular Myocytes to H2O2 Increases the Level of Mitochondrial NADH. We have detected an upregulation of TCA cycle proteins succinyl-CoA and malate dehydrogenase after exposure of myocytes to H2O2 using iTRAQ analysis (Table 2). The TCA cycle produces NADH and FADH2 that are required for oxidative phosphorylation and production of ATP. We therefore examined whether a transient exposure to H2O2 can alter production of mitochondrial NADH. Myocytes were exposed to 30 µM H2O2 for 5 min followed by 10 units/ mL catalase for 5 min, and NADH autofluorescence was assessed. In eight cells, exposure to H2O2 resulted in a 10.5 ( 2.3% increase in the magnitude of the NADH signal compared to cells exposed to 0 µM H2O2 for 5 min followed by 10 units/ mL catalase [n ) 8; p < 0.05 (Figure 4)]. Addition of 20 µM oligomycin further increased the magnitude of the NADH signal, consistent with an active TCA cycle, and NADH fluorescence decreased after addition of FCCP, consistent with depletion of NADH after collapse of the mitochondrial membrane potential.20,25 The TCA cycle enzyme R-ketoglutarate dehydrogenase inhibitor R-keto-β-methyl-n-valeric acid (KMV) attenuated the increase in the magnitude of the NADH signal
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Altered Ventricular Cardiomyocyte Protein Expression and Growth Table 3. Proteins Identified as Downregulated by iTRAQ Analysis protein name
mitochondrial, TCA cycle citrate synthase mitochondrial, oxidative phosphorylation cytochrome c oxidase subunit 2 mitochondrial, other functions ubiquinone biosynthesis protein COQ9, mitochondrial precursor aspartate aminotransferase, mitochondrial precursor sulfite oxidase, mitochondrial precursor prohibitin-2 ubiquinone biosynthesis protein COQ7 homologue glycine cleavage system H protein cytoplasmic myoglobin sarcolemmal membrane-associated protein peptidyl-prolyl cis-trans isomerase FKBP1A protein NipSnap homologue 2 nuclear splicing factor, arginine/serine-rich 4 miscellaneous hemoglobin β-subunit hemoglobin R-subunit (hemoglobin R-chain)
% coverage
no. of peptidesa
30.7
accession numberb
117:115c
Unused Prot Scored
p valuee
17
13435
0.8322
23.66
0.0055
15.0
4
14211
0.8859
5.03
0.0229
25.7
7
04099
0.9075
9.29
0.0086
63.0 25.6 30.4 29.8 17.3
40 11 6 5 2
08228 08936 18339 09621 08959
0.8568 0.8802 0.9342 0.8846 0.8447
56.89 15.53 5.88 5.33 4.00
0 0.0417 0.0478 0.0101 0.0397
61.7 18.7 46.3 19.9
14 13 3 7
06188 17400 02937 04106
0.8795 0.8897 0.7126 0.8871
23.62 16.37 6.03 3.01
0.0264 0.0295 0.0428 0.0311
23.4
7
06543
0.9186
5.86
0.0171
68.0 49.3
10 9
01378 00235
0.4666 0.4712
21.16 14.81
0 0
a Number of unique peptide sequences assigned to the protein. b All accession numbers were prefaced by ENSCPOP000000. c 117:115 is the average protein ratio of cells treated with 30 µM H2O2 for 5 min and then 10 units/mL catalase to cells treated with 0 µM H2O2 for 5 min and then 10 units/mL catalase. d The Unused Prot Score is a measure of all the peptide evidence for a protein that is not better explained by a higher-ranking protein. For proteins to be identified with >95%, the required Unused Prot Score is 1.3. e Proteins were only considered to be statistically significantly up- or downregulated if p < 0.05.
attenuated the increase in metabolic activity (Figure 5). Our iTRAQ analysis identified alterations in the expression of mitochondrial proteins, but it does not provide information regarding protein activity. The increase in mitochondrial metabolic activity measured 48 h after transient exposure to H2O2 suggests the increase in the level of protein expression correlated with increased activity, thereby validating the alterations in protein expression identified with iTRAQ analysis.
Discussion Figure 3. Pie chart demonstrating the cellular distribution of all proteins with altered expression identified by iTRAQ analysis according to the PANTHER classification system.
(Figure 4). These results indicate that a transient exposure of myocytes to H2O2 increases the level of mitochondrial NADH production as a result of an increase in TCA cycle enzyme activity. Transient Exposure of Ventricular Myocytes to H2O2 Increases Mitochondrial Metabolic Activity. Our iTRAQ analysis identified alterations in the expression of a number of proteins involved in oxidative phosphorylation (Tables 2 and 3). Consequently, we examined whether a transient exposure of myocytes to H2O2 altered oxygen consumption and metabolic activity in the myocytes. We assessed metabolic activity as the formation of formazan from tetrazolium in intact myocytes. 48 h after exposure of myocytes to 30 µM H2O2 for 5 min followed by 10 units/mL catalase for 5 min, metabolic activity was significantly increased (Figure 5). Consistent with an increase in the level of intracellular calcium mediating the increase in metabolic activity,16,21 application of the ICa-L antagonist nisoldipine prior to addition of H2O2 and preventing the uptake of calcium into the mitochondria with Ru360
The early mechanisms for the development of pathological hypertrophy in the ischemic heart remain poorly understood. Since a transient exposure of ventricular myocytes to H2O2 can result in persistent activation of ICa-L, an increase in the diastolic calcium level, and a further increase in mitochondrially derived superoxide without apoptosis or necrosis,16 we speculated that this could represent an early mechanism for the induction of cardiac hypertrophy. We have combined a proteomics/iTRAQbased screening approach with validation by a biological assay to investigate this. Our results indicate that a 5 min exposure of cardiac myocytes to 30 µM H2O2 is sufficient to increase the total level of protein synthesis assessed as incorporation of [3H]leucine 48 h later (Figure 1). Given the antioxidant capacity of the myocyte, 30 µM H2O2 equates to approximately 3 µM intracellular H2O2, which is higher than physiological signaling levels and considered to represent mild oxidative stress.2 Consistent with an increase in calcium influx through ICa-L mediating protein synthesis, nisoldipine but not dantrolene (an inhibitor of RyR calcium release) attenuated the response. In addition, a Ca2+-calmoldulin-dependent signaling pathway appeared to be involved because KN-62 attenuated the increase in the rate of incorporation of [3H]leucine (Figure 1). Our results indicate that an increase in the diastolic calcium level as a result of increased calcium influx through ICa-L is sufficient to activate Journal of Proteome Research • Vol. 9, No. 4, 2010 1991
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Figure 4. Transient exposure to H2O2 increases the level of NADH. (A) NADH fluorescence recorded from a myocyte before and after exposure to 0 µM H2O2 for 5 min followed by 10 units/mL catalase (Cat) for 5 min, from a myocyte before and after exposure to 30 µM H2O2 for 5 min followed by 10 units/mL catalase for 5 min, and from another myocyte following addition of 10 mM KMV and then 30 µM H2O2 for 5 min followed by 10 units/mL catalase for 5 min. The arrow indicates when additions were made. Twenty micromolar oligomycin (Oligo) and 4 µM FCCP were added where indicated (see the text for an explanation). (B) Mean ( SEM of the percent increase in NADH fluorescence for myocytes exposed to 0 µM H2O2 for 5 min followed by 10 units/ mL catalase (Cat) for 5 min or 30 µM H2O2 for 5 min followed by 10 units/mL catalase for 5 min or 10 mM KMV as indicated.
Ca2+-dependent growth pathways that then increase the level of protein synthesis in ventricular myocytes. The activation of signaling pathways occurs within 48 h of transient exposure to H2O2 and can be reversed when calcium influx through ICa-L is prevented with nisoldipine. Additional characteristics of hypertrophy include an increase in cell size and disruption of cytoskeletal proteins. We identified a small but significant increase in cell size (Figure 2A), but no evidence of major actin cytoskeleton disorganization that is typically observed in advanced cardiac hypertrophy when cells were stained with phalloidin or desmin (Figure 2B,C). This is probably because we were looking for evidence of cytoskeletal disruption at an early stage in the hypertrophic process. We propose that more marked disarray would become evident as the hypertrophy progresses over time. A total of 669 unique proteins were successfully identified as mapping to the myocytes of guinea pig. Consistent with an increase in the total protein level (Figure 1), we identified alterations in expression of 35 proteins using iTRAQ analysis. Of the 35 proteins, five sarcomeric proteins were upregulated (Table 2). Given the abundance of the sarcomeric proteins in 1992
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Figure 5. Transient exposure to H2O2 increases metabolic activity in cardiac myocytes. (A) MTT assay performed in myocytes following exposure to 0 µM H2O2 for 5 min followed by 10 units/ mL catalase (Cat) for 5 min, 30 µM H2O2 for 5 min followed by 10 units/mL catalase for 5 min, 2 µM nisoldipine (Nisol) and then 30 µM H2O2 for 5 min followed by 10 units/mL catalase for 5 min, or 2 µM Ru360 and then 30 µM H2O2 for 5 min followed by 10 units/mL catalase for 5 min. Slopes represent changes in absorbance at 570 nm (with a reference wavelength of 620 nm) measured over 0-1 min. (B) Means ( SEM of the percent increase in absorbance measured over 0-1 min for myocytes exposed to treatments as indicated compared to cells not treated (control).
cardiac myocytes, the increase in the level of expression of the proteins could contribute to the increase in cell size we observed. Consistent with this, we identified a decrease in the level of expression of prohibitin. Although prohibitin assumes multiple roles in the mitochondria, including a chaperone for newly synthesized respiratory complexes in the inner mitochondrial membrane, its function includes the control of cell proliferation.26 The downregulation of prohibitin has been associated with increased cell growth and may contribute to the increase in cell size measured 48 h after exposure to H2O2 (Figure 2A). The majority of proteins with altered expression were mitochondrial in origin and included proteins that regulate function in the mitochondrial respiratory chain or energy metabolism (Tables 2 and 3). Exposure to 30 µM H2O2 significantly increased NADH (Figure 4) and metabolic activity (Figure 5) in the myocytes. The increase in metabolic activity was attenuated with prior exposure of cells to the ICa-L antagonist nisoldipine and preventing the uptake of calcium into the mitochondria with Ru360 (Figure 5). We have shown previously that an increase in calcium influx, as a result of direct activation of ICa-L with Bay K 8644, is sufficient to increase mitochondrial
Altered Ventricular Cardiomyocyte Protein Expression and Growth 21
NADH and metabolic activity. An increase in TCA cycle activity and NADH production can also in theory increase the rate of electron flow down the respiratory chain and contribute to the increased level of superoxide production at Complex I or III of the mitochondria.27 These sequelae are consistent with the persistent increase in the level of superoxide we observe after transient exposure of myocytes to H2O2 that can be attenuated with Ru360 or with direct block of ICa-L with nisoldipine.16 The increase for superoxide is not due to increased NADPH-oxidase, xanthine oxidase, or nitric oxide activity.16 These alternative sources of superoxide do not appear to contribute during an acute increase in oxidative stress. Our results suggest that a persistent alteration of calcium homeostasis after activation of the L-type Ca2+ channel by H2O2 is sufficient to induce cardiac hypertrophy. An increase in the intracellular calcium level has been implicated in the development of cardiac hypertrophy through activation of signaling pathways such as ERK, MAPK, and NFAT.6-9 A persistent increase in the diastolic calcium level appears to be sufficient for the induction of oxidative stress in the myocyte due to an increased level of activation of TCA cycle enzymes and increased superoxide production by the mitochondria. Therefore, changes in mitochondrial metabolic activity may be important in the early pathophysiological mechanisms associated with the development of cardiac hypertrophy in ischemic heart disease.
Conclusions It is well-established that oxidative stress is a feature of cardiac hypertrophy and cardiac failure. Using iTRAQ protein labeling followed by mass spectrometry analysis (LC-MALDIMSMS), we identified altered expression of 35 proteins after transient exposure of myocytes to H2O2. The majority of the altered proteins were mitochondrial, consistent with the persistent increase in the level of superoxide measured after exposure of the myocytes to H2O2. Our results suggest that transient oxidative stress participates early in the induction of cardiac hypertrophy, and increased calcium influx through ICa-L contributes to an increase in the level of protein synthesis and alterations in expression and function of mitochondrial proteins. Mitochondrial dysfunction and poor energy metabolism are well recognized in the ischemic and failing myocardium, but the pathophysiology is poorly understood.13,28 This study has provided insight into early mechanisms for induction of hypertrophy involving increased oxidative stress and intracellular calcium and the development of mitochondrial dysfunction that is a common feature of cardiac disease. Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; ICa-L, L-type Ca2+ channel; TCA, tricarboxylic acid; HBS, HEPES-buffered saline; NFAT, calcineurin/nuclear factor of activated T cells; CaMK, calcium calmodulin-dependent protein kinases; ROS, reactive oxygen species; RyR, ryanodine receptor; KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]4-phenylpiperazine, a specific CaMK inhibitor; FCCP, carbonyl cyanide-(trifluoromethoxy)phenyl-hydrazone; FDR, false discovery rate.
Acknowledgment. This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC). V.S. is a recipient of a Western Australia State Government Science and Innovation Studentship Award. H.M.V. is a recipient of a Biomedical Postgraduate Research Scholarship from NHMRC and the National Heart
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Foundation of Australia. G.R. is supported by NHMRC Project Grant 403941. T.M.C. is a recipient of funding from the WA Centre for Food and Genomic Medicine. E.I. is a recipient of funding from NHMRC (Grants 513714 and 403987) and the Cancer Council of Western Australia. N.G.L. is a recipient of NHMRC Principal Research Fellowship 403904. L.C.H. is a recipient of NHMRC Career Development Award 303225. The mass spectrometry analyses were performed in facilities provided by the Lotterywest State Biomedical Facility-Proteomics Node, Western Australian Institute for Medical Research.
Supporting Information Available: Full list of 669 peptides identified by the iTRAQ approach (Table S1) and peptide assignments for all proteins identified (Table S2). This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Bergamini, C. M.; Gambetti, S.; Dondi, A.; Cervellati, C. Oxygen, reactive oxygen species and tissue damage. Curr. Pharm. Des. 2004, 10, 1611–1626. (2) Stone, J. R.; Yang, S. Hydrogen peroxide: A signaling messenger. Antioxid. Redox Signaling 2006, 8, 243–270. (3) Becker, L. B. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc. Res. 2004, 61, 461–470. (4) Dhalla, N. S.; Temsah, R. M.; Netticadan, T. Role of oxidative stress in cardiovascular diseases. J. Hypertens. 2000, 18, 655–673. (5) Wolin, M. S. Interactions of oxidants with vascular signaling systems. Arterioscler., Thromb., Vasc. Biol. 2000, 20, 1430–1442. (6) van Empel, V. P.; De Windt, L. J. Myocyte hypertrophy and apoptosis: A balancing act. Cardiovasc. Res. 2004, 63, 487–499. (7) Frey, N.; Olson, E. N. Cardiac hypertrophy: The good, the bad, and the ugly. Annu. Rev. Physiol. 2003, 65, 45–79. (8) Wilkins, B. J.; Molkentin, J. D. Calcium-calcineurin signaling in the regulation of cardiac hypertrophy. Biochem. Biophys. Res. Commun. 2004, 322, 1178–1191. (9) Dorn, G. W., II; Force, T. Protein kinase cascades in the regulation of cardiac hypertrophy. J. Clin. Invest. 2005, 115, 527–537. (10) Tomida, T.; Hirose, K.; Takizawa, A.; Shibasaki, F.; Iino, M. NFAT functions as a working memory of Ca2+ signals in decoding Ca2+ oscillation. EMBO J. 2003, 22, 3825–3832. (11) Dolmetsch, R. E.; Lewis, R. S.; Goodnow, C. C.; Healy, J. I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 1997, 386, 855–858. (12) Molkentin, J. D.; Lu, J. R.; Antos, C. L.; Markham, B.; Richardson, J.; Robbins, J.; Grant, S. R.; Olson, E. N. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 1998, 93, 215–228. (13) Lesnefsky, E. J.; Moghaddas, S.; Tandler, B.; Kerner, J.; Hoppel, C. L. Mitochondrial dysfunction in cardiac disease: Ischemiareperfusion, aging, and heart failure. J. Mol. Cell. Cardiol. 2001, 33, 1065–1089. (14) Zorov, D. B.; Filburn, C. R.; Klotz, L. O.; Zweier, J. L.; Sollott, S. J. Reactive oxygen species (ROS)-induced ROS release: A new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J. Exp. Med. 2000, 192, 1001–1014. (15) Aon, M. A.; Cortassa, S.; Marban, E.; O’Rourke, B. Synchronized whole cell oscillations in mitochondrial metabolism triggered by a local release of reactive oxygen species in cardiac myocytes. J. Biol. Chem. 2003, 278, 44735–44744. (16) Viola, H. M.; Arthur, P. G.; Hool, L. C. A transient exposure to hydrogen peroxide causes an increase in mitochondrial-derived superoxide as a result of sustained alteration in L-type Ca2+ channel function in the absence of apoptosis in ventricular myocytes. Circ. Res. 2007, 100, 1036–1044. (17) Rosati, B.; Dong, M.; Cheng, L.; Liou, S. R.; Yan, Q.; Park, J. Y.; Shiang, E.; Sanguinetti, M.; Wang, H. S.; McKinnon, D. Evolution of ventricular myocyte electrophysiology. Physiol. Genomics 2008, 35, 262–272. (18) Hool, L. C. Hypoxia increases the sensitivity of the L-type Ca2+ current to β-adrenergic receptor stimulation via a C2 regioncontaining protein kinase C isoform. Circ. Res. 2000, 87, 1164– 1171.
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PR9011393
