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Toxicology 260 (2009) 84–96

Contents lists available at ScienceDirect

Toxicology

journa l homepage: www.e lsev ier .com/ locate / tox ico l

drenaline and reactive oxygen species elicit proteome and energeticetabolism modifications in freshly isolated rat cardiomyocytes

era Marisa Costaa,∗, Renata Silvaa, Ludgero Canário Tavaresb, Rui Vitorinoc,rancisco Amadoc, Félix Carvalhoa, Maria de Lourdes Bastosa, Márcia Carvalhoa,d,ui Albuquerque Carvalhob, Fernando Remiãoa,∗

REQUIMTE (Rede de Química e Tecnologia), Toxicology Department, Faculty of Pharmacy, University of Porto, Porto, PortugalNeurosciences Center of Coimbra, University of Coimbra, Coimbra, PortugalChemistry Department, University of Aveiro, Aveiro, PortugalCEBIMED, Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal

r t i c l e i n f o

rticle history:eceived 17 February 2009ccepted 15 March 2009vailable online 25 March 2009

eywords:roteomicsardiomyocytesitochondria

drenalineeactive oxygen species

a b s t r a c t

The sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), andthe generation of reactive oxygen species (ROS) are well recognized hallmarks of several cardiopatho-logic conditions, like cardiac ischemia/reperfusion (I/R) and heart failure (HF). The present work aimed toinvestigate the proteomics and energetic metabolism of cardiomyocytes incubated with ADR and/or ROS.To mimic pathologic conditions, freshly isolated calcium-tolerant cardiomyocytes from adult rat wereincubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xan-thine oxidase) (XXO)]. Two-dimensional electrophoresis with matrix-assisted laser desorption/ionizationand time-of-flight mass spectrometer analysis were used to define protein spot alterations in the car-diomyocytes incubated with ADR and/or ROS. Moreover, the energetic metabolism and the activity ofmitochondrial complexes were evaluated by nuclear magnetic resonance and spectrophotometric deter-minations, respectively. The protein extract was mainly constituted by cardiac mitochondrial proteinsand the alterations found were included in five functional classes: (i) structural proteins, notably myosinlight chain-2; (ii) redox regulation proteins, in particular superoxide dismutase (SOD); (iii) energeticmetabolism proteins, encompassing ATP synthase alpha chain and dihydrolipoyllysine-residue acetyl-transferase component of pyruvate dehydrogenase complex; (iv) stress response proteins, like the heatshock proteins; and (v) regulatory proteins, like cytochrome c and voltage-dependent anion channel 1.The XXO system elicited alterations in cardiac contractile proteins, as they showed high levels of cleavage,and also altered energetic metabolism, through increased lactate and alanine levels. The cardiomyocytes

incubation with ADR resulted in an accentuated increase in mitochondrial complexes activity and thedecrease in alanine/lactate ratio, thus reflecting a high cytosolic NADH/NAD+ ratio. Furthermore, anincrease in manganese SOD expression and total SOD activity occurred in the ADR group, as the increasein the mitochondrial complexes presumably led to higher ‘electron leakage’. The modifications in pro-teins, enzymes activity, and energetic metabolism were indicative that different pathways are activatedby catecholamines and ROS. Thcontribute for the initiation or

Abbreviations: I/R, ischemia/reperfusion injury; HF, heart failure; ROS, reac-ive oxygen species; ADR, adrenaline; XXO, xanthine and xanthine oxidase; TTC,,3,5-triphenyltetrazolium chloride; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyletrazolium bromide; PDH, pyruvate dehydrogenase; MALDI, matrix-assistedaser desorption/ionization; TOF, time-of-flight mass spectrometer; 2-DGE, two-imensional gel electrophoresis; PTM, post transcriptional modifications; NMR,uclear magnetic resonance; MW, molecular weight; pI, isoelectric point; HSP,eat shock protein; SOD, superoxide dismutase; VDAC-1, voltage-dependent anionhannel-1; LDH, lactate dehydrogenase; ETC, electron transport chain.∗ Corresponding authors. Tel.: +351 222078979; fax: +351 222003977.

E-mail addresses: veramcosta@ff.up.pt (V.M. Costa), remiao@ff.up.pt (F. Remião).

300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2009.03.012

ese alterations altogether determine the I/R and HF specific features andaggravation of those cardiopathologic conditions.

© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Stressful stimuli often lead to strenuous release of adrenaline(ADR) and noradrenaline throughout the nervous system andadrenal medulla (Behonick et al., 2001). Elevated concentrations ofcirculating and/or interstitial myocardial catecholamines are alsofound in the event of arrhythmias (Malhotra et al., 2002), myocar-

dial necrosis (Behonick et al., 2001; Lameris et al., 2000), heartfailure (HF) (Hasking et al., 1986; Johansson et al., 1997), exercise(Kjaer, 1998), pheochromocytoma (Gerlo and Sevens, 1994), hypo-glycaemia, hemorrhagic hypotension, circulatory collapse, distress(Goldstein et al., 2003), and in the ischemic and reperfusion (I/R)

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njury (Behonick et al., 2001; Killingsworth et al., 2004; Lamerist al., 2000). In the I/R and HF, oxidative stress is also a recog-ized hallmark (Zweier and Talukder, 2006). In fact, in the ischemicnd post-ischemic myocardium, reactive oxygen species (ROS) areormed at an accelerated rate (Killingsworth et al., 2004; Lamerist al., 2000) and it is now accepted that many of the deleteri-us cellular phenotypes present in the hypertrophied and failingyocardium may be attributed to oxidative stress (Ferrari et al.,

998; Kaye and Esler, 2005; Kinugawa et al., 2000; McMurrayt al., 1993; Sawyer et al., 2002). Although the toxicity of cate-holamines has been mainly attributed to continuous stimulation ofdrenoceptors, there is an increasing evidence that the oxidation ofhese molecules is also responsible for cardiotoxicity (Bindoli et al.,992; Costa et al., 2009a,b, 2007; Dhalla et al., 2001; Remião et al.,001a, 2002, 2004). Thus, high levels of ROS and catecholamines aressociated with several detrimental effects in heart, whose patho-hysiological features need to be further clarified.

Cardiac proteins are in a dynamic state of continuous degrada-ion and resynthesis, thus any alteration in protein turnover playsn important role in normal heart muscle homeostasis (Doll et al.,007). Several stimuli can impair that equilibrium. Hypertrophyue to excessively stimulated protein synthesis is induced in adultardiomyocytes under pathophysiological conditions (Schluter etl., 1995). Also, the protein content in the cardiomyocytes can beltered in result of compromised protein degradation (Schluter etl., 1995). The ubiquitin-proteasome system is the main machinerynvolved in the non-lysosomal degradation of short-lived, damagednd misfolded intracellular proteins in eukaryotic cells (Nandi et al.,006). Consequently, the modification of the ubiquitin-proteasomeystem disrupts the cardiac protein homeostasis (Doll et al., 2007).

Under pathological conditions several proteins pools can beltered, namely those involved in the energetic metabolism. Gly-olytic and oxidative metabolism of exogenous glucose can bexamined coordinately by measuring the levels of glucose, glyco-en and its metabolic intermediaries, namely pyruvate, lactate orlanine (Carvalho et al., 2004a; McNulty et al., 2000). Mitochon-ria are responsible for the majority of ATP production in the cellsnd they have a key role in the signalling cascades involved inrogrammed cell death (Lesnefsky et al., 2001). Also, mitochon-ria are a major source and target of ROS (Lesnefsky et al., 2001).or those reasons, mitochondria are a major target in toxicitytudies.

The present study was devoted to the evaluation of the pro-ein turnover in cardiomyocytes under the influence of ADR and ofROS generating system (xanthine with xanthine oxidase—XXO).

n previous works, protein modifications and proteasome impair-ent in cardiomyocytes incubated with ADR and ADR with XXOere observed (Costa et al., 2007, 2009b). In the present study,

wo-dimensional gel electrophoresis (2-DGE) and matrix-assistedaser desorption/ionization (MALDI) with time-of-flight (TOF) masspectrometer were used for a wide proteome analysis. Further-ore, the activity of several mitochondrial complexes and the

lucose metabolism were evaluated through spectrophotometriceasurements and nuclear magnetic resonance (NMR), respec-

ively. Possible correlations between the proteomics data andnergetic metabolism in the different treatments were also eval-ated.

. Materials and methods

.1. Animals

Adult male Sprague Dawley rats (Charles—River Laboratories, Barcelona, Spain)eighing 250–350 g were used. The animals were housed in cages with temperature-

nd humidity-controlled environment. Food and water were provided ad libitum andnimals were subjected to a 12 h light/dark cycle. Animal experiments were licensedy the Portuguese General Directory of Veterinary Medicine. Housing and experi-ental treatment of the animals were in accordance with the Guide for the Care and

y 260 (2009) 84–96 85

Use of Laboratory Animals from the Institute for Laboratory Animal Research (ILAR1996). The experiments complied with Portuguese current laws.

2.2. Chemicals

All reagents used in this study were of analytical grade. Collagenase typeII was obtained from Worthington (Lakewood, New Jersey, USA). Collagenase(type IA), bovine serum albumin (fraction V), N-(2-hydroxyethyl) piperazine-N-(2-ethanesulfonic acid) (HEPES), phenylmethylsulfonyl (PMSF), ADR, xanthine,xanthine oxidase, dithiothreitol (DTT), aprotinin, leupeptin, pepsatin, ethylene gly-col tetraacetic acid (EGTA), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazoliumbromide (MTT), 2,3,5-triphenyltetrazolium chloride (TTC), and sodium fumaratewere purchased to Sigma–Aldrich (St. Louis, MO, USA). Immobillines pH 3–11, IPGstrips (13 cm pH 3–11), and all general reagents used for the MALDI analysis werepurchased to Amersham Pharmacia Biotech Europe (Uppsala, Sweden). Trypsinwas from Promega (Madison, WI, USA), �-cyano-4-hydroxycinnamic acid and thecalibrant mixture for the 4800 Proteomics Analyzer were obtained from AppliedBiosystems (Foster City, CA, USA).

2.3. Isolation of calcium-tolerant cardiomyocytes from adult rat

Calcium-tolerant cardiomyocytes were isolated by Langendorff retro-perfusionof adult rat heart as previously described (Costa et al., 2007; Remião et al., 2001b,2002). At the beginning of the experiments, cell viability was always greater than60%, as evaluated by the lactate dehydrogenase (LDH) leakage assay and by micro-scopic evaluation of cardiomyocytes morphology. The obtained viability was inaccordance with previous reports for calcium-tolerant cardiomyocytes (Carvalho etal., 2004b; Cordeiro et al., 1994; Costa et al., 2009a,b; Remião et al., 2004). Thisviability was obtained after a 5 min pre-incubation period at 37 ◦C to guarantythe correspondence between values obtained in manual counting and LDH leak-age assay. Incubations were performed in a water bath at 37 ◦C, using a density of2.5 × 105 viable cells/mL in the modified Krebs–Henseleit buffer supplemented with1 mM CaCl2 (pH 7.4) and saturated with an airstream of carbogen, every hour. Afterthe pre-incubation period, the compounds were tested for 3 h using the followingprotocol: (i) control cells, with no exposure; (ii) cells incubated with ADR alone;(iii) cells exposed to ADR with XXO; and (iv) cells exposed only to XXO. The finalconcentrations were: 500 �M ADR, 100 �M xanthine and 0.01 U/mL XO.

2.4. Extraction of mitochondrial proteins in cardiomyocytes

In the mitochondrial protein extraction of cardiomyocytes, we tried to elimi-nate most of the cytoplasmic proteins (Costa et al., 2009b). An aliquot of the cellsuspension was collected and centrifuged for 10 min at 2000 × g. Supernatant wasdiscarded, and to the pellet was added 1 mL of AC solution [(in mM): 10 HEPES (pH7.9), 10 KCl, 1.5 MgCl2, 0.5 EDTA, 0.1 EGTA plus 0.1% Igepal], supplemented with 1 mMDTT and 0.25 mM PMSF. After vortexing for a few seconds, cells were incubated onice for 15 min and then centrifuged for 10 min at 2000 × g, at 4 ◦C. The supernatantwas rejected. To obtain an enriched mitochondrial fraction, further extraction wasperformed. To the previous obtained pellet was added 50–60 �L of BC solution [(inmM: 20 HEPES (pH 7.9); 420 NaCl; 1.5 MgCl2; 0.5 disodium EDTA; 1 DTT, and 0.25PMSF), 20% glycerol, and 5 �g/mL each of the following protease inhibitors: apro-tinin, leupeptin, pepsatin]. The samples were placed in ultrasonic bath for 15 minand then incubated on ice for 30 min. After centrifugation, at 16,000 × g for 25 min,the supernatant was used for 2-DGE gel and MALDI-TOF-TOF analysis. This crudeextract is mainly rich in cardiac mitochondrial proteins.

2.5. Two-dimensional gel electrophoresis (2-DGE)

2-DGE analysis of cardiomyocytes crude protein extracts was performed in a hor-izontal apparatus (IPGphor and Hoefer 600 SE from Amersham Pharmacia Biotech).Briefly, 270 �g of protein were applied onto IPG strips (13 cm, pH 3–11), contain-ing immobilines pH 3–11, thiourea, CHAPS, and urea (Vitorino et al., 2004). Afterisoelectric focusing, the strip was applied on top of a SDS-polyacrylamide (12.5%)electrophoresis (SDS-PAGE) gel (12 cm/614 cm, 12.5%) and proteins were separatedaccording to their molecular weight (MW). The SDS-PAGE gel was stained with col-loidal coomassie blue and the 2-DGE maps were analyzed with PDQuest Software(Bio-Rad). Optical density results were expressed as fold increase over control values.Results were obtained from 2 different gels for each treatment (with cardiomyocytesuspensions obtained from 2 different rats).

2.6. Tryptic digestion

For tryptic digestion, the spots of 2-DGE were removed and digested by trypsin to

be identified by MALDI-TOF-TOF. The protocol used for tryptic digestion was accord-ing to Vitorino et al. (2004). The protein spots were excised with a pipette tip from thegel and transferred to the Investigator ProGest automated digester (Genomic Solu-tions, Ann Arbor, MI, USA) rack. The gel pieces were washed three times with 25 mMammonium bicarbonate/50% acetonitrile (ACN), one time with ACN and dried ina SpeedVac (Thermo Savant); 25 �L of 10 �g/mL sequence grade modified porcine

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rypsin (Promega) in 25 mM ammonium bicarbonate was added to the dried gelieces and the samples were incubated overnight at 37 ◦C.

.7. Peptide identification by MALDI-TOF-TOF

Extraction of tryptic peptides was performed by adding 10% of formic acidFA)/50% ACN three times and lyophilized in a SpeedVac. Tryptic peptides were resus-ended in 10 �L of a 50% ACN/0.1% FA solution. The samples were mixed (1:1) withmatrix consisting of a saturated solution of �-cyano-4-hydroxycinnamic acid pre-ared in 50% ACN/0.1% FA. Aliquots of samples (0.5 �L) were spotted onto the MALDIample target plate. Peptide mass spectra were obtained on a MALDI-TOF-TOF masspectrometer (4800 Proteomics Analyzer; Applied Biosystems) in the positive ioneflector mode. Every day, and before an automated acquisition, two spots weresed for signal and parameter optimization. Spectra were obtained in the mass rangeetween 800 and 4000 Da with ca. 1500 laser shots. For each sample spot, a data-ependent acquisition method was created to select the two most intense peaks,xcluding those from the matrix, due to trypsin autolysis, or acrylamide peaks, forubsequent MS/MS data acquisition. Trypsin autolysis peaks were used for internalalibration of the mass spectra, allowing a routine mass accuracy of better than5 ppm. The control of ion fragmentation energy was achieved by changing theragmentation gas type used for MS/MS spectra acquisition.

.8. Database search

Spectra were processed and analyzed by the Global Protein Server WorkstationApplied Biosystems), which uses internal Mascot (Matrix Science Ltd., UK) softwareor searching the peptide mass fingerprints and MS/MS data. Searches were per-ormed against the NCBI non-redundant protein database. Further confirmation ofrotein identifications was obtained by duplicating the protein identifications usingrotein Prospector (www.prospector.ucsf.edu, from the University of California atan Francisco) and/or Prowl (www.prowl.rockfeller.com, Rockefeller University atew York Universities) software.

.9. Determination of glucose, lactate and alanine levels in cardiomyocyteuspensions

The levels of glucose, lactate and alanine were determined by proton nuclearagnetic resonance (1H NMR) in the supernatant of cardiomyocyte suspensions.

ontrol, ADR, ADR with XXO, and XXO cells were centrifuged for 2 min at 18 × g andhe supernatant was removed and immediately frozen. Each NMR sample consistedf 160 �L of supernatant and 40 �L of 2 mM sodium fumarate solution in D2O, useds an internal standard. 1H NMR spectra were acquired in a 600 MHz Varian Spec-rometer equipped with a 3 mm inverse detection NMR probe. Spectrum acquisitionarameters included a 45◦ radiofrequency excitation pulse, a 3 s acquisition time24k points covering a sweep width of 8 kHz), and a pre-acquisition delay of 17 s,hich included a 3 s water saturation delay, for allowing complete nuclei relaxation

or quantification purposes. Each spectrum consisted of a total of 32 accumulationso achieve adequate signal to noise. A 0.2 Hz exponential multiplication function andero filling were applied to the free induction decay (FID) before Fourier transforma-ion using the NutsProTM (NMR Utility Transform Software—Professional, Acorn NMRnc., Freemont, USA). Metabolite concentration was made by spectral deconvolutionf the glucose-H1� (doublet at ı = 5.22 ppm), lactate-CH3 (doublet at ı = 1.33 ppm)nd alanine-CH3 (doublet at ı = 1.48 ppm) resonances and subsequent comparisonith the fumarate resonance (singlet at ı = 6.50 ppm).

.10. Evaluation of mitochondrial function through the methylthiazoleetrazolium (MTT) and 2,3,5-triphenyltetrazolium chloride (TTC) assays

Mitochondrial function was evaluated spectrophotometrically by measuring theegree of mitochondrial reduction of the tetrazolium salts, MTT and TTC, to for-azans. Samples (800 �L) of cell suspensions from the 4 treatment groups were

entrifuged at 18 × g for 2 min to separate the cells. The cells were washed twicey adding 1 mL of modified Krebs–Henseleit buffer supplemented with 1 mM CaCl2ollowed by centrifugation at 18 × g for 2 min. The obtained washing solutions wereejected and cells were resuspended in 200 �L of fresh modified Krebs–Henseleituffer supplemented with 1 mM CaCl2 (Costa et al., 2007).

In the MTT test, 20 �L of 5 mg MTT/mL were added to the washed cells. Cells werencubated at 37 ◦C for 30 min and the reaction was stopped by addition of 200 �L 10%odium dodecyl sulfate (SDS) in 0.01 M hydrochloric acid. An overnight incubationt 37 ◦C was required for the formazan solubilisation and the photometric detectionf the blue formazan was performed at 550 nm (Capela et al., 2007).

For the TTC determination, 800 �L of cell suspensions were redrawn after the 3 h

ncubation, washed twice, and resuspended in 400 �L of modified Krebs–Henseleituffer supplemented with 1 mM CaCl2, as described above. After adding 16 �L of0 mg TTC/mL and incubating at 37 ◦C for 3 h, the reaction was stopped with 800 �L0% SDS in 0.01 M hydrochloric acid, which was followed by overnight incubationt 37 ◦C and photometric detection of the respective carmine formazan at 490 nmDong et al., 1997).

y 260 (2009) 84–96

2.11. Determination of total superoxide dismutase (SOD) activity

The SOD determination was performed after centrifugation of cell suspensionsat 18 × g for 2 min, for separation of supernatant and pellet, as previously described(Costa et al., 2007). The pellet was placed at −80 ◦C for further analysis.

A xanthine–xanthine oxidase system was used to generate superoxide radicals,and the subsequent reduction of nitroblue tetrazolium (NBT) by those radicals wasmonitored at 560 nm. SOD activity was expressed as units per milligram of protein(one unit of SOD is defined as the amount of enzyme required to inhibit the rate ofNBT reduction by 50%) (Flohé and Otting, 1984).

2.12. Lactate dehydrogenase (LDH) leakage assay

LDH leakage assay was performed in the cardiomyocyte suspensions to evaluatethe level of cell injury at time 0 h (immediately after addition of the compounds) andafter 3 h incubation in all treatments, as previously described (Costa et al., 2009b).

2.13. Protein determination

Protein content was determined by the method described by Bradford (1976),using bovine serum albumin as standard.

2.14. Statistical analysis

Results obtained from lactate and glucose levels, MTT and TTC reduction aregiven as mean ± standard deviation (SD) from 5 to 6 experiments (with cardiomy-ocyte suspensions obtained from 5 to 6 different rats). Non-parametric tests wereused. Statistical comparisons between groups were performed by Kruskal–Wallistest (one-way ANOVA on ranks) followed by the Student–Newman-Keuls post hoctest, once a significant p had been obtained (p < 0.05).

3. Results

3.1. The proteomics of freshly isolated cardiomyocytes from adultrat

After the 3 h incubation period, in the control group, 103 pro-tein spots were separated and identified after 2-DGE (Table 1 ).In accordance to the protein isolation procedure, most of the pro-teins found and identified in the extract were from mitochondria,although it was not a pure extract (Fig. 1). In the control group,several of the spots showed redundancy, as they were identifiedas the same protein with some alterations when compared to thetheoretical characteristics of the native proteins. The majority ofthe altered protein spots were reported to: (i) isoelectric point (pI)shift; or (ii) significantly different MW when compared to the nativeprotein. Those changes can be attributed to the cardiomyocytes iso-lation procedure and are summarized in Table 2. Explicitly, spot1 showed changed pI compared to native cytochrome c (spot 98).Spots 3 and 4 had lower MW than myosin regulatory light chain2 (spot 5). Spot 6 had higher MW than the native translationallycontrolled tumor protein. Spot 9 had a lower pI than expected inacetyl-CoA acetyltransferase. Spot 14 had higher MW than spot11 (myosin light polypeptide 3). Spot 17 had lower MW than thenative desmin. Spots 20 and 22 had higher MW than spot 38 (fattyacid-binding protein), while spot 26 had lower pI and spot 56 hadhigher pI. Spot 25 had lower pI than the native cytochrome c oxidasepolypeptide Vb. Spots 39 and 55 had lower MW than 37 (myosinheavy chain). Spot 41 had a lower pI than spot 57 (thioredoxin-dependent peroxidase reductase). Spot 42 had a lower MW thanheat shock cognate 71 kDa. Spot 48 had higher MW than ATP syn-thase D chain mitochondrial. Spot 50 had lower pI and higherMW than 72 (enoyl-CoA hydratase). Spot 51 had lower pI thandihydrolipoyllysine-residue succinyltransferase of 2-oxoglutaratedehydrogenase complex. Spot 52 had higher MW than myosin lightpolypeptide (spot 11). Spot 59 had a lower MW than spot 79 (aconi-

tase hydratase). Spot 68 has lower MW than myosin heavy chain,cardiac muscle beta isoform. Spot 97 had higher pI than spot 82(myoglobin). Spot 89 had a lower MW than spot 102 (isocitratedehydrogenase). Spot 94 had a higher MW than native alpha eno-lase. Spot 81 had a lower pI than spot 96 (manganese SOD). Both

V.M. Costa et al. / Toxicology 260 (2009) 84–96 87

Table 1Table of spots identified in isolated cardiomyocytes from adult rats by MALDI-TOF-TOF, with peptide count and protein score.

Spot no. Identification Accession no./species Theoretical mass/pI Peptide count Protein score

1 Cytochrome c, somatic P62898/Rat 11.4/9.60 6 2192 Troponin C P19123/Mouse 18.4/4.04 6 1703 Myosin regulatory light chain 2 P08733/Mouse 18.7/4.71 11 2754 Myosin regulatory light chain 2 P08733/Mouse 18.7/4,71 4 1325 Myosin regulatory light chain 2 P08733/Rat 18.7/4,86 10 1776 Translationally controlled tumor protein P63029/Rat 19.4/4.76 6 1247 Calmodulin P62161/Rat 17.0/4.09 3 1268 Complement component 1, Q subcomponent binding protein P63029/Rat 30.7/4.81 3 1259 Acetyl-CoA acetyltransferase P17764/Rat 44.7/8.92 12 180

10 Tropomyosin 1 alpha chain P04692/Rat 32.7/4.71 28 74411 Myosin light polypeptide 3 P16409/Rat 22.0/5.03 11 45112 Myosin regulatory light chain 2 P08733/Rat 18.7/4.86 9 41013 Cytochrome c oxidase polypeptide Va P11240/Rat 16.1/6.08 8 22114 Myosin light polypeptide 3 P16409/Rat 22.0/5.03 13 42315 NADH-ubiquinone oxidoreductase 23 kDa subunit 8K3J1/Mouse 24.0/5.89 7 15916 Peroxiredoxin 2 P35704/Rat 21.8/5.34 3 11317 Desmin P48675/Rat 53.3/5.21 5 13918 Actin, alpha cardiac P68035/Rat 42.0/5.23 10 32719 ATP synthase beta chain P10719/Rat 56.3/5.19 24 60720 Fatty acid-binding protein, heart P07483/Rat 14.6/5.92 3 11521 Serine/threonine protein phosphatase 2A, 65 kDa regulatory

subunit A, alpha isoformQ76MZ3/Mouse 65.2/5.00 4 63

22 Fatty acid-binding protein, heart P07483/Rat 14.6/5.92 3 11823 Heat shock protein HSP90-beta P34058/Rat 83.1/5.06 6 12124 78 kDa glucose-regulated protein precursor P20029/Mouse 72.4/5.07 24 63425 Cytochrome c oxidase polypeptide Vb P12075/Rat 13.9/7.68 4 13526 Fatty acid-binding protein, heart P07483/Rat 14.6/5.92 5 28627 Heat-shock protein beta-2 O35878/Rat 20.3/5.27 4 8428 Pyruvate dehydrogenase E1 component beta subunit P49432/Rat 38.8/5.94 13 20829 Troponin T, cardiac muscle isoform P50753/Rat 35.6/4.95 9 22930 Ubiquinol-cytochrome c reductase complex core protein I Q9CZ13/Mouse 52.7/5.75 9 10531 Heat shock cognate 71 kDa protein P63018/Rat 70.8/5.24 23 33932 60 kDa heat shock protein P63039/Rat 60.9/5.91 9 20533 Transitional endoplasmic reticulum ATPase P46462/Rat 89.3/5.14 23 30934 NADH-ubiquinone oxidoreductase 75 kDa subunit Q91VD9/Mouse 79.7/5.51 11 14235 Alpha-actinin 1 Q9Z1P2/Rat 103/5.23 13 7836 Stress-70 protein P48721/Rat 73.8/5.97 8 16537 Myosin heavy chain, cardiac muscle alpha isoform P02563/Rat 223/5.59 41 50838 Fatty acid-binding protein, heart P07483/Rat 14.6/5.92 8 22339 Myosin heavy chain, cardiac muscle alpha isoform P02563/Rat 223/5.59 7 10640 Peroxiredoxin 6 O35244/Rat 24.7/5.65 7 9741 Thioredoxin-dependent peroxide reductase P20108/Mouse 28.1/7.15 2 9042 Heat shock cognate 71 kDa protein P63018/Rat 70.8/5.37 18 22343 NADH-ubiquinone oxidoreductase 30 kDa subunit Q9DCT2/Mouse 30.2/6.40 10 28144 L-lactate dehydrogenase B chain P42123/Rat 36.5/5.17 17 35345 Malate dehydrogenase, cytoplasmic P14152/Mouse 36.3/6.16 6 14546 Prohibitin P67779/Rat 29.8/5.17 8 24447 Succinyl-CoA ligase [GDP-forming] beta-chain Q9Z2I8/Mouse 43.7/5.75 8 21248 ATP synthase D chain, mitochondrial P31399/Rat 18.6/6.21 3 8349 Succinyl-CoA ligase [ADP-forming] beta-chain Q9Z2I9/Mouse 50.1/6.57 8 16550 Enoyl-CoA hydratase P14604/Rat 31.5/8.39 8 9651 Dihydrolipoyllysine-residue succinyltransferase of 2-oxoglutarate

dehydrogenase complexQ01205/Rat 47.4/8.17 5 137

52 Myosin light polypeptide 3 P16409/Rat 22.0/5.03 12 22353 Dihydrolipoyllysine-residue acetyltransferase component of

pyruvate dehydrogenase complexP08461/Rat 58.7/5.70 6 145

54 Superoxide dismutase [Cu-Zn] P07632/Rat 15.8/5.89 6 17755 Myosin heavy chain, cardiac muscle alpha isoform P02563/Rat 223/5.59 18 17256 Fatty acid-binding protein, heart P07483/Rat 14.6/5.92 5 16057 Thioredoxin-dependent peroxide reductase P20108/Mouse 28.1/7.15 9 17658 Heat-shock protein beta-6 P97541/Rat 17.5/6.05 4 6559 Aconitate hydratase Q9ER34/Rat 85.4/7.87 14 13160 Acyl-CoA dehydrogenase, long-chain specific P15650/Rat 47.8/7.63 18 28561 Isovaleryl-CoA dehydrogenase P12007/Rat 46.4/8.03 11 21762 Acyl-CoA dehydrogenase, long-chain specific P15650/Rat 47.8/7.63 18 28563 Alpha enolase P04764/Rat 46.7/6.16 8 8864 NADH-ubiquinone oxidoreductase 49 kDa subunit Q91WD5/Mouse 52.6/6.52 8 6665 Aldehyde dehydrogenase P11884/Rat 56.5/6.63 8 23366 Succinate dehydrogenase flavoprotein subunit Q920L2/Rat 71.6/6.75 13 25567 Propionyl-CoA carboxylase alpha chain P14882/Rat 77.7/6.33 18 19768 Myosin heavy chain, cardiac muscle beta isoform P02564/Rat 223/5.64 5 5669 NADH-ubiquinone oxidoreductase 13 kDa-B subunit Q63362/Rat 13.2/7.07 3 5670 Alpha crystallin B chain P2392/Rat 20.1/6.76 8 12071 Delta3,5-delta2,4-dienoyl-CoA isomerase Q62651/Rat 36.1/8.13 10 31872 Enoyl-CoA hydratase, P14604/Rat 31.5/8.39 10 20173 Creatine kinase, M chain P00564/Rat 43.0/9.58 9 286

88 V.M. Costa et al. / Toxicology 260 (2009) 84–96

Table 1 (Continued )

Spot no. Identification Accession no./species Theoretical mass/pI Peptide count Protein score

74 Electron transfer flavoprotein-ubiquinone oxidoreductase Q6UPE1/Rat 68.2/7.33 13 11575 Carnitine O-palmitoyltransferase II P18886/Rat 74.0/6.89 7 8676 Dihydrolipoyl dehydrogenase O08749/Mouse 54.2/7.97 9 10277 ATP synthase alpha chain P15999/Rat 58.8/9.22 11 9678 Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 Q9D0K2/Rat 55.6/8.73 9 7179 Aconitate hydratase Q9ER34/Rat 85.4/7.87 25 28780 Nucleoside diphosphate kinase B P19804/Rat 17.2/6.92 8 19881 Superoxide dismutase [Mn] P07895/Rat 24.7/8.96 8 13082 Myoglobin P04248/NE 16.9/7.27 4 8083 Fatty acid-binding protein, adipocyte P70623/Rat 14.7/7.93 2 9184 Electron transfer flavoprotein alpha-subunit P13803/Rat 35.0/8.67 10 12885 Adenylate kinase isoenzyme 2 Q9WTP6/Mouse 25.5/7.16 4 8386 Electron transfer flavoprotein beta-subunit Q9DCW4/Mouse 27.3/8.57 4 8687 Creatine kinase P09605/Rat 47.4/8.76 14 24588 Aspartate aminotransferase, cytoplasmic P13221/Rat 46.7/6.3 15 18589 Isocitrate dehydrogenase [NADP] P54071/Mouse 58.7/8.89 6 9190 Beta enolase P15429/Rat 46.8/7.74 4 10791 Fumarate hydratase P14408/Rat 54.4/9.06 11 14492 Glutamate dehydrogenase 1 P10860/Rat 61.4/8.05 12 17993 Pyruvate kinase, isozymes M1/M2 P11980/Rat 57.7/6.69 16 14394 Alpha enolase P04764/Rat 46.7/6.16 5 11195 Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 Q9D0K2/Mouse 55.6/8.73 16 14096 Superoxide dismutase [Mn] P07895/Rat 24.7/8.96 8 7797 Myoglobin P04247/Mouse 16.9/7.23 3 8598 Cytochrome c, somatic P62898/Rat 11.5/9.61 5 27199 Short chain 3-hydroxyacyl-CoA dehydrogenase Q9WVK7/Rat 34.4/8.83 7 129

100 Voltage-dependent anion-selective channel protein 1 Q9Z2L0/Rat 30.6/8.63 6 119101 Malate dehydrogenase P08249/Rat 35.6/8.92 14 366102 Isocitrate dehydrogenase [NADP] P54071/Mouse 58.7/8.89 15 344103 ATP synthase alpha chain P15999/Rat 58.8/9.22 17 282

A andw incubm

sc

3X

ts

ccession numbers in the Swiss-Prot along with animal species, theoretical massere analyzed from cardiomyocytes providing from two different animals after 3 husculus); NE (Nannospalax ehrenbergi).

pots 77 and 103 had lower pI than the native ATP synthase alphahain.

.2. Cardiomyocyte incubation with adrenaline, adrenaline with

XO, and XXO resulted in protein alterations

The ratio between the optic densities of the spots of differentreatments (ADR, ADR with XXO and XXO) to control was taken, andeveral spots were found to be altered (Table 3). The majority of the

Fig. 1. Image of a two-dimensional gel, stained with colloidal coomassie blue, of mitoc

isoelectric point (pI) are also briefly summarized in the table. Two different gelsation. Protein score C.I.% always 100%. Species: Rat (Rattus norvegicus); Mouse (Mus

altered protein spots at 3 h showed different abundance, possiblyindicating that the initial protein was either modified to another pIvariant, proteolyzed to leave fragments that remained undetectedin the system analysis, polymerized or with reduced expression.

The spots abundance of the structural proteins was highlyaltered in the treated groups. An increase in the myosin regula-tory light chain 2 (spot 5) and of some of its fragments (spot 3) inboth ADR with XXO, and XXO alone was observed (Table 3). Thelevels of myosin light polypeptide 3 (spot 11) and troponin T (spot

hondrial enriched protein extract of control cardiomyocytes after 3 h incubation.

V.M. Costa et al. / Toxicology 260 (2009) 84–96 89

Table 2Comparative table showing the spots analyzed by MALDI-TOF-TOF in control cardiomyocytes after 3 h incubation whose values of molecular weight (MW) and isoelectricpoint (pI) differ from the theoretical ones, according to Swiss-Prot.

Spot no. Identification Theoretical mass/pI Modification Native protein

1 Cytochrome c, somatic 11.4/9.60 Lower pI Spot 983 Myosin regulatory light chain 2 18.7/4.71 Lower MW Spot 54 Myosin regulatory light chain 2 18.7/4,71 Lower MW9 Acetyl-CoA acetyltransferase 44.7/8.92 Lower pI

13 Cytochrome c oxidase polypeptide Va 16.1/6.08 Lower pI14 Myosin light polypeptide 3 22.0/5.03 Higher MW Spot 1117 Desmin 53.3/5.21 Lower MW20 Fatty acid-binding protein, heart 14.6/5.92 Higher MW Spot 3822 Fatty acid-binding protein, heart 14.6/5.92 Higher MW Spot 3825 Cytochrome c oxidase polypeptide Vb 13.9/7.68 Lower pI26 Fatty acid-binding protein, heart 14.6/5.92 Lower pI Spot 3839 Myosin heavy chain, cardiac muscle alpha isoform 223/5.59 Lower MW Spot 3741 Thioredoxin-dependent peroxide reductase 28.1/7.15 Lower pI Spot 5742 Heat shock cognate 71 kDa protein 70.8/5.37 Lower MW48 ATP synthase D chain, mitochondrial 18.6/6.21 Higher MW50 Enoyl-CoA hydratase 31.5/8.39 Lower pI and higher MW Spot 7251 Dihydrolipoyllysine-residue succinyltransferase of

2-oxoglutarate dehydrogenase complex47.4/8.17 Lower pI

52 Myosin light polypeptide 3 22.0/5.03 Higher MW and pI Spot 1155 Myosin heavy chain, cardiac muscle alpha isoform 223/5.59 Lower MW Spot 3756 Fatty acid-binding protein, heart 14.6/5.92 Higher MW Spot 3859 Aconitate hydratase 85.4/7.87 Lower MW Spot 7968 Myosin heavy chain, cardiac muscle beta isoform 223/5.64 Lower MW and higher pI77 ATP synthase alpha chain 58.8/9.22 Lower pI81 Superoxide dismutase [Mn] 24.7/8.96 Lower pI Spot 9689 Isocitrate dehydrogenase [NADP] 58.7/8.89 Lower MW Spot 102

415

2iaa43

ihtitIk(taea4htwr(g5iakad

wgw

94 Alpha enolase97 Myoglobin

103 ATP synthase alpha chain

9) increased in XXO group. Alpha-actinin 1 (spot 35) increasedn the ADR group. The abundance of other structural proteins wasltered, as observed in ADR, ADR with XXO, and XXO groups. Thebundance of the fragment of myosin regulatory light chain 2 (spot), of desmin (spot 17) and of the altered myosin light polypeptide(spot 14) decreased when compared with control.

Proteins of energetic metabolism suffered modifications dur-ng the different treatments (Table 3). The ADR with XXO groupad a notorious decrease in the relative abundance of electronransfer flavoprotein beta-subunit (spot 86) and of pyruvate kinase,sozymes M1/M2 (spot 93). The ADR group showed an increase inhe abundance of ATP synthase alpha chain (spots 77 and 103).n the XXO group, creatine kinase, M chain (spot 73), pyruvateinase, isozymes M1/M2 (spot 93) and ATP synthase alpha chainspot 103) significantly decreased. In particular, most of the pro-eins involved in the carbohydrate metabolism (native proteinsnd altered proteins) had an abundance decrease in XXO, with thexception of fumarase hydratase (spot 91). In the ADR group, thebundance of succinyl-CoA ligase [GDP-forming] beta-chain (spot7), fumarate hydratase (spot 91) and of the fragment of aconitaseydratase (spot 59) increased slightly, while the fragments of isoci-rate dehydrogenase [NADP] (spots 89 and 102) decreased. The ADRith XXO group had an accentuate decrease in dihydrolipoyllysine-

esidue acetyltransferase component of pyruvate dehydrogenasePDH) complex (spot 53) and of the fragment of isocitrate dehydro-enase [NADP] (spot 89). The abundance of several proteins (spots0, 71, 72, and 99) involved in the fatty acid metabolism decreased

n XXO. In the ADR with XXO group, the abundance in the fattycid metabolism proteins decreased in spots 50, 72, and 99. Inetone metabolism proteins, acetyl-CoA acetyltransferase (spot 9)nd succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (spot 78)

ecreased significantly in XXO.

The levels of redox regulatory proteins changed in ADR, ADRith XXO, and XXO groups when compared to control. In the ADR

roup, the SODs (Mn and Cu-Zn) (spots 54, 81 and 96) increased,hile peroxiredoxin 6 (spot 40) decreased. In the ADR with XXO, the

6.7/6.16 Higher MW6.9/7.23 Higher pI Spot 828.8/9.22 Lower pI

peroxiredoxin 6 (spot 40) and MnSOD (spot 96) decreased, whileCu-ZnSOD (spot 54) increased.

The abundance of stress response proteins was different in thetreatment groups. In ADR with XXO, the abundance of 78 kDaglucose-regulated protein precursor (spot 24) increased while theabundance of stress-70 protein (spot 36), heat shock protein (HSP)90-beta (spot 23), and HSP beta-6 (spot 58) decreased. The ADRgroup had an increased abundance in the fragment of heat shockcognate 71 kDa protein (spot 42) in the crude protein extract, whileHSP beta-6 (spot 58) decreased. In XXO group, a noticeable decreasein HSP 90-beta (spot 23), 60 kDa heat shock protein (spot 32), heatshock cognate 71 kDa protein (spot 42), HSP beta-6 (spot 58), andalpha crystallin B chain (spot 70) was observed in the mitochondrialcrude extract.

In the crude extract, cytochrome c (spot 98) rose in all treatmentgroups, while the pI altered cytochrome c (spot 1) largely increasedin ADR and ADR with XXO. In ADR and ADR with XXO groups, thecomplement component 1, Q subcomponent binding protein (spot8) and prohibitin (spot 46) increased while serine/threonine pro-tein phosphatase 2A, 65 kDa regulatory subunit A, alpha isoform(spot 21) and adenylate kinase isoenzyme 2 (spot 85) decreased.The fatty acid-binding protein apparently suffered several posttranscriptional modifications (PTM). The polymerized fatty acid-binding protein in spot 20 decreased in all treatment groups, whilethe pI altered protein (spot 26) increased in the ADR group and thenative protein (spot 38) decreased slightly in XXO when comparedto control. Voltage-dependent anion-selective channel protein 1(VDAC-1) (spot 100) increased in ADR and ADR with XXO groupswhen compared to control.

3.3. The incubation with adrenaline, adrenaline with XXO and

XXO disrupted the overall glycolytic pathway

Fig. 2 shows expansions of representative 1H NMR spectraobtained in the four experimental groups: control, ADR, ADRwith XXO, and XXO at 3 h; focusing the resonances of fumarate

90 V.M. Costa et al. / Toxicology 260 (2009) 84–96

Table 3Optic density of spots after incubation with adrenaline 500 �M (ADR), adrenaline 500 �M with xanthine and xanthine oxidase (ADR with XXO), and XXO relative to control(set to 1).

Spot no. Identification ADR ADR with X/XO X/XO

(a) Sarcomeric and cytoskeletal proteins3 Myosin regulatory light chain 2 1.9 ± 0.3 10.7 ± 1.2 17.4 ± 0.114 Myosin regulatory light chain 2 0.7 ± 0.09 0.3 ± 0.3 0.6 ± 0.25 Myosin regulatory light chain 2 0.6 ± 0.7 10.0 ± 2.5 24.9 ± 3.7

11 Myosin light polypeptide 3 0.6 ± 0.3 1.3 ± 0.4 5.4 ± 0.314 Myosin light polypeptide 3 0.6 ± 0.1 0.02 ± 0.02 0.2 ± 0.117 Desmin 0.2 ± 0.4 0.3 ± 0.5 0.4 ± 0.529 Troponin T, cardiac muscle isoform 1.2 ± 0.3 1.0 ± 0.5 3.4 ± 1.035 Alpha-actinin 1 10.5 ± 0.9 1.0 ± 0.5 0.6 ± 0.8

(b) MetabolismATP synthesis

73 Creatine kinase, M chain 1.1 ± 0.4 0.8 ± 0.7 0.5 ± 0.0577 ATP synthase alpha chain 1.9 ± 0.4 0.9 ± 0.5 1.4 ± 0.486 Electron transfer flavoprotein beta-subunit 0.7 ± 0.5 0.002 ± 0.2 1.0 ± 0.587 Creatine kinase 0.5 ± 0.4 0.6 ± 0.5 0.6 ± 0.593 Pyruvate kinase, isozymes M1/M2 0.7 ± 0.1 0.5 ± 0.03 0.4 ± 0.2

103 ATP synthase alpha chain 3.3 ± 0.5 1.1 ± 0.5 0.3 ± 0.4

Carbohydrate metabolism44 L-lactate dehydrogenase B chain 1.1 ± 0.04 0.8 ± 0.03 0.5 ± 0.245 Malate dehydrogenase, cytoplasmic 1.1 ± 0.01 0.8 ± 0.05 0.6 ± 0.0447 Succinyl-CoA ligase [GDP-forming] beta-chain 1.6 ± 0.4 0.8 ± 0.04 0.6 ± 0.0853 Dihydrolipoyllysine-residue acetyltransferase component of

pyruvate dehydrogenase complex0.7 ± 0.5 0.2 ± 0.2 0.1 ± 0.3

59 Aconitate hydratase 1.4 ± 0.5 0.9 ± 0.5 0.3 ± 0.589 Isocitrate dehydrogenase [NADP] 0.2 ± 0.3 0.07 ± 0.1 0.6 ± 0.591 Fumarate hydratase 1.5 ± 0.4 1.2 ± 0.5 2.3 ± 0.594 Alpha enolase 0.5 ± 0.5 0.4 ± 0.2 0.3 ± 0.3

101 Malate dehydrogenase 1.2 ± 0.5 0.5 ± 0.5 0.2 ± 0.4102 Isocitrate dehydrogenase [NADP] 0.3 ± 0.09 0.6 ± 0.5 0.5 ± 0.04

Fatty acid metabolism50 Enoyl-CoA hydratase 0.4 ± 0.3 0.2 ± 0.4 0.5 ± 0.571 Delta 3,5-delta2,4-dienoyl-CoA isomerase 1.2 ± 0.4 0.9 ± 0.2 0.5 ± 0.0272 Enoyl-CoA hydratase 0.6 ± 0.4 0.4 ± 0.0 0.5 ± 0.0199 Short chain 3-hydroxyacyl-CoA dehydrogenase 1.0 ± 0.5 0.3 ± 0.3 0.3 ± 0.3

Ketone metabolism9 Acetyl-CoA acetyltransferase 0.5 ± 0.03 0.7 ± 0.1 0.3 ± 0.03

78 Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 0.8 ± 0.5 0.5 ± 0.4 0.08 ± 0.5

(c) Redox regulatory proteins40 Peroxiredoxin 6 0.5 ± 0.3 0.2 ± 0.2 0.6 ± 0.554 Superoxide dismutase [Cu-Zn] 2.0 ± 0.8 2.0 ± 0.9 1.0 ± 1.081 Superoxide dismutase [Mn] 2.8 ± 0.2 1.1 ± 0.5 0.9 ± 0.596 Superoxide dismutase [Mn] 1.5 ± 0.5 0.4 ± 0.3 0.6 ± 0.5

(d) Stress response proteins23 Heat shock protein HSP90-beta 1.3 ± 0.8 0.4 ± 0.5 0.4 ± 0.524 78 kDa glucose-regulated protein precursor 1.3 ± 0.09 1.6 ± 0.2 0.8 ± 0.232 60 kDa heat shock protein 1.3 ± 0.2 1.0 ± 0.02 0.4 ± 0.136 Stress-70 protein 0.7 ± 0.1 0.4 ± 0.09 0.5 ± 0.642 Heat shock cognate 71 kDa protein 1.9 ± 0.5 0.7 ± 0.5 0.5 ± 0.558 Heat-shock protein beta-6 0.3 ± 0.5 0.0 0.070 Alpha crystallin B chain 1.0 ± 0.1 0.9 ± 0.03 0.5 ± 0.2

(e) Regulatory proteins1 Cytochrome c, somatic 1.5 ± 0.8 1.9 ± 0.2 0.5 ± 0.28 Complement component 1, Q subcomponent binding protein 1.7 ± 0.7 2.1 ± 0.7 1.0 ± 0.2

20 Fatty acid-binding protein, heart 0.6 ± 0.02 0.5 ± 0.2 0.6 ± 0.0121 Serine/threonine protein phosphatase 2A, 65 kDa regulatory

subunit A, alpha isoform0.07 ± 0.1 0.3 ± 0.4 0.9 ± 0.4

22 Fatty acid-binding protein, heart 0.2 ± 0.2 0.2 ± 0.1 1.9 ± 0.426 Fatty acid-binding protein, heart 1.6 ± 0.6 1 ± 0.6 0.7 ± 0.638 Fatty acid-binding protein, heart 0.9 ± 0.04 0.8 ± 0.0 0.5 ± 0.246 Prohibitin 1.5 ± 0.4 1.6 ± 0.4 0.5 ± 0.585 Adenylate kinase isoenzyme 2 0.09 ± 0.2 0.01 ± 0.2 0.8 ± 0.5

T nimal

(bts

98 Cytochrome c, somatic100 Voltage-dependent anion-selective channel protein 1

wo different gels were analyzed providing from cardiomyocytes of two different a

CH CH ; a), used as internal standard, and of glucose (H1�;), alanine ( CH3; c) and lactate ( CH3; d) used for determina-ion of the concentration of such metabolites in the cardiomyocyteuspension media.

2.6 ± 0.3 3.7 ± 0.3 3.4 ± 0.32.2 ± 0.5 1.3 ± 0.4 0.2 ± 0.4

s after 3 h incubation with the different treatments. Control set to 1.

In Fig. 3A, the glucose levels in the supernatant of the cardiomy-ocyte suspensions in control, ADR, ADR with XXO, and XXO areshown during the 3 h incubation. During the study, the levels ofglucose in control group were steady. At 3 h, a significant decrease

V.M. Costa et al. / Toxicolog

Fig. 2. Expansions from fumarate (a, ı = 5.50 ppm), glucose-H1� (b, ı = 5.22 ppm),ar53

i((

t(ilXh

ocrXnmgt

icwfip

lanine –CH3 (c, ı = 1.48 ppm) and lactate –CH3 (d, ı = 1.33 ppm) resonances fromepresentative 1H NMR spectra of control (C), adrenaline 500 �M (ADR), adrenaline00 �M with xanthine and xanthine oxidase (ADR with XXO), and XXO groups afterh incubation.

n the levels of glucose in ADR (8.2 ± 1.7 mM) and ADR with XXO8.2 ± 1.0 mM) groups was observed when compared to control11.6 ± 1.3 mM).

During the present study, the levels of lactate were assessed inhe supernatant of control, ADR, ADR with XXO, and XXO groupsFig. 3B). The lactate levels in all treatment groups rose signif-cantly when compared to control, as soon as 1 h. At 3 h, theactate values in all treatment groups [ADR (3.8 ± 1.8 mM), ADR withXO (5.3 ± 0.9 mM) and XXO (5.1 ± 1.0 mM)] remained significantlyigher than control (2.8 ± 1.0 mM).

In Fig. 3C, the alanine levels in the supernatant of the cardiomy-cyte suspensions in control, ADR, ADR with XXO, and XXO groupsan be observed during the 3 h study. The alanine values in controlose steadily along the study. At 1 h, the alanine values in ADR withXO (0.28 ± 0.04 mM) and XXO (0.31 ± 0.07 mM) groups were sig-ificantly higher than control (0.19 ± 0.03 mM). This tendency wasaintained in the other incubation time points. At 2 h, the ADR

roup had significantly lower values of alanine (0.19 ± 0.03 mM)han control (0.29 ± 0.07 mM).

Fig. 3D shows the ratio between alanine/lactate levels in 5ndependent experiments. The ratio in XXO group showed signifi-

ant differences at 2 h (0.14 ± 0.03) and 3 h (0.21 ± 0.05) incubationhen compared to control (C = 2, 0.11 ± 0.04; C = 3, 0.16 ± 0.06),

avouring the alanine formation. At 3 h, the ratio of alanine/lactaten the ADR group significantly decreased (0.11 ± 0.06) when com-ared to control (0.16 ± 0.06).

y 260 (2009) 84–96 91

3.4. MTT and TTC reduction was increased in adrenaline treatedcardiomyocytes

At 3 h, the activity of mitochondrial complexes was evaluatedthrough the formation of formazans in the washed cardiomyocytes.MTT reduction is shown in Fig. 4A and is related with NADH dehy-drogenase (complex I) and/or succinate dehydrogenase (complexII) activities (Dong et al., 1997). TTC reduction (Fig. 4B) is relatedwith the activity of the cytochrome c oxidase (complex IV) (Donget al., 1997).

At 3 h, the MTT metabolized in the ADR group increased signif-icantly (218 ± 64%) when compared to control (100%) (Fig. 4A). At3 h, in the ADR with XXO group, MTT rose to 162 ± 30% and XXOgroup showed no differences to control.

After 3 h, the TTC was significantly higher in the ADR group(168 ± 25%) when compared to control (100%) (Fig. 4B), while theADR with XXO, and XXO groups showed no significant differencesto control.

3.5. Total SOD activity increased after exposure to adrenaline butdecreased in the presence of XXO

The activity of total SOD was evaluated after the 3 h incubationperiod (Fig. 5). The activity of SOD in the ADR group significantlyincreased (21.1 ± 2.9 units/mg of protein) when compared to control(16.9 ± 2.3 units/mg of protein). In the ADR with XXO group, theactivity of total SOD decreased to 10.6 ± 2.9 units/mg of protein andin the XXO group to 9.3 ± 2.5 units/mg of protein, significantly lowerthan control (16.9 ± 2.3 units/mg of protein).

3.6. Incubation with adrenaline, adrenaline with XXO, and XXOdid not affect cardiomyocytes viability

After the 3 h incubation, none of the treated cell groups (exposedto ADR, ADR with XXO, and XXO) showed any change in cells viabil-ity when compared to the control group by the LDH leakage assay(data not shown).

4. Discussion

Despite years of research, the mechanisms of myocardial tox-icity induced by catecholamines and oxidative stress are not yetfully clarified. Through a 2-DGE approach, subtle and unsus-pected changes in the mitochondrial extract of cardiac myocytesexposed to ADR, XXO (ROS generating system), or ADR with XXOwere detected. The results provided further insights towards theelicited intracellular mechanisms since several proteins showedPTM and/or altered abundance. This work showed that the expo-sure to ADR or to XXO revealed different proteomic and metabolicalterations. The main protein alterations can be classified as fol-lows: (i) pI shifts (away from the predicted pI of the protein); (ii)significantly lower or higher MW than predicted, indicative of par-tial loss of the native protein or polymerization, respectively; and(iii) altered abundance (indicative that the native protein was eithermodified to another pI, altered in expression, PTM or proteolyzedto fragments that remained undetectable in the analysis system).Of the 61 altered spots found in the present work (Table 3), themajority of changes were observed in proteins of five functionalgroups: (i) structural proteins of the sarcomere and cytoskeleton,notably myosin light chain-2, myosin light polypeptide 3, alpha-actinin, and troponin T; (ii) proteins of the energetic metabolism,

outstandingly, ATP synthase alpha chain and dihydrolipoyllysine-residue acetyltransferase component of PDH complex; (iii) redoxregulation proteins, in particular SOD; (iv) stress response proteins,namely heat shock proteins (HSP); and (v) regulatory proteins, ascytochrome c and VDAC-1.

92 V.M. Costa et al. / Toxicology 260 (2009) 84–96

Fig. 3. (A) Levels of glucose (mM) in the supernatant of cardiomyocyte suspensions in control (C), adrenaline (ADR) 500 �M, adrenaline 500 �M with xanthine and xanthineoxidase (ADR with XXO), and XXO groups during the 3 h incubation time. Means ± SD, n = 6, Kruskal–Wallis test, followed by the Student–Newman–Keuls post hoc test(**p < 0.01 vs. control). (B) Levels of lactate (mM) in the supernatant of cardiomyocyte suspensions in control (C), adrenaline 500 �M (ADR), adrenaline 500 �M with xanthineand xanthine oxidase (ADR with XXO), and XXO groups during the 3 h incubation time. Means ± SD, n = 5, Kruskal–Wallis test, followed by the Student–Newman–Keuls posthoc test (*p < 0.05 vs. control). (C) Levels of alanine (mM) in the supernatant of cardiomyocyte suspensions in control (C), adrenaline 500 �M (ADR), adrenaline 500 �M withx ubatiop 0 h).s thineM t hoc t

ernptwarle

ttof(cppw

uoracbt5PId

anthine and xanthine oxidase (ADR with XXO), and XXO groups during the 3 h incost hoc test (*p < 0.05 vs. control; **p < 0.01 vs. control; ##p < 0.01 vs. control timeuspensions in control (C), adrenaline (ADR) 500 �M, adrenaline 500 �M with xaneans ± SD, n = 5, Kruskal–Wallis test, followed by the Student–Newman–Keuls pos

Modifications in the sarcomeric proteins of the cardiomyocytesxposed to ADR with XXO or to XXO alone, in particular myosinegulatory light chain 2, were probably the result of cleavage of theative protein, but can also reveal compromised quality control ofroteins. In fact, in previous works, we demonstrated an early pro-easome activity inhibition in rat cardiomyocytes exposed to ADRith XXO or to XXO (Costa et al., 2009b) with accumulation of dam-

ged, oxidized and/or misfolded proteins (Costa et al., 2007). Ouresults confirm previous reports showing that oxidative stressors,ike those formed in I/R in the heart, alter myofibrillar proteins andlicit contractile dysfunction (White et al., 2005, 2006).

Glucose metabolism for energy generation in the heart requireshe coordinated action of transmembrane transport, phosphoryla-ion, glycolysis, and subsequent tricarboxylic acid cycle oxidationf glycolytic pyruvate (McNulty et al., 2000). The main substratesor mitochondrial oxidation in the heart are carbohydrates and fatsStanley et al., 2005), while in freshly isolated cardiomyocytes, glu-ose is the main substrate (Remião et al., 2001b). The glycolyticathway results in the formation of cytosolic NADH and, in theresent work that was favoured in ADR and ADR with XXO groupsith concomitant decrease in glucose levels (Fig. 3A).

Pyruvate resulting from glycolysis is a target for mitochondrialptake, or can be metabolized in the cytosol by the reversiblexidation to lactate, via LDH (Lesnefsky et al., 2001) or theeversible transamination by alanine transaminase (ALT) to formlanine (Fischer et al., 1997). Pyruvate metabolism in the mito-hondria requires the uptake by the pyruvate transporter followedy oxidation by PDH to acetyl-CoA. In the present work, of all

he subunits of PDH identified by MALDI-TOF-TOF, only spot3, dihydrolipoyllysine-residue acetyltransferase component ofDH complex, showed differences between treatments (Table 3).n fact, in ADR with XXO, and XXO groups, the abundance ofihydrolipoyllysine-residue acetyltransferase component of PDH

n. Means ± SD, n = 6, Kruskal–Wallis test, followed by the Student–Newman–Keuls(D) Ratio between alanine and lactate levels in the supernatant of cardiomyocyteand xanthine oxidase (ADR with XXO), and XXO groups during the 3 h incubation.est (*p < 0.05 vs. control; **p < 0.01 vs. control; #p < 0.05 vs. control time 0 h).

complex was greatly reduced, possibly resulting of PTM. Also, LDHin the XXO group was greatly decreased (Table 3). The XXO grouphad the highest levels of lactate and alanine at 3 h and, as they arebyproducts of cytosolic pyruvate, the cytosolic pyruvate accumu-lation can be assumed to have occurred. Furthermore, pyruvateand lactate are known to inhibit glucose transport while alanineincreases glucose transport (Fischer et al., 1997, 1990). Thus, in XXOgroup, the balance between lactate, alanine and pyruvate levelselicited low glucose utilization and possibly inhibited its inwardtransport (Fig. 3A). In fact, in XXO group the alanine formationwas favoured (Fig. 3C) and the ratio alanine/lactate was the high-est (Fig. 3D), reflecting a low NADH/NAD+ ratio (thus low glycolyticactivity) and the decrease in the cytosolic redox status (Yu et al.,1996).

During the mitochondrial metabolism, the pyruvate derivedacetyl-CoA is oxidized completely to CO2 in the tricarboxylic acidcycle. The electrons from these oxidations are transferred to O2through a chain of carriers (electron transport chain—ETC) in themitochondrion leading to the formation of ATP and H2O (Lesnefskyet al., 2001). The modifications observed in the present work inthe ATP synthase alpha subunit, a protein subunit of ATP syn-thase (complex V) were of particular interest (Table 3). The twoATP synthase alpha subunit spots (spots 77 and 103) showed a pIshift that may be the result of PTM, namely phosphorylation(s).Both spots of ATP synthase alpha subunit increased in the ADRgroup, therefore indicating that the ETC was altered in this group.The total ATP content in all treatment groups did not change afterthe 3 h of incubation, as observed in a previous work (Costa et al.,

2007). However, the dissimilarities in the glucose and lactate val-ues (Fig. 3A and B) and in TTC and MTT reduction (Fig. 4A and B)reveal that the different treatments elicited different metabolic pro-files in the cardiomyocytes. The low alanine/lactate ratio in the ADRgroup suggests an increase in cytosolic redox state (NADH/NAD+),

V.M. Costa et al. / Toxicolog

Fig. 4. (A) MTT reduction in cardiomyocytes exposed to adrenaline 500 �M (ADR),adrenaline 500 �M with xanthine and xanthine oxidase (ADR with XXO), and XXOalone at 3 h. Results are presented as means ± SD from 6 different experimentswhen compared with control, which was set to 100%. Statistical comparisons weremade using Kruskal–Wallis test, followed by the Student–Newman–Keuls post hoctest (**p < 0.01 vs. control). (B) TTC reduction in cardiomyocytes after 3 h incubationwith adrenaline 500 �M (ADR), adrenaline 500 �M with xanthine and xanthine oxi-dase (ADR with XXO), and XXO alone at 3 h. Results are presented as means ± SDf1S

pctfemasdpoiobtp

atmsF(io

group resulted in increased chaperon activity of HSP70 towardscytoplasmatic proteins, whereas in the ADR with XXO group, theHSP70 family related response was wider (Table 3). In contrast,the ADR group showed a substantial increase in the fragments of

Fig. 5. Total superoxide dismutase activity (units/mg protein) in cardiomyocytes

rom 6 different experiments when compared with control (C), which was set to00%. Statistical comparisons were made using Kruskal–Wallis test, followed by thetudent–Newman–Keuls post hoc test (**p < 0.01 vs. control).

resumably due to high glycolytic activity (Fig. 3D). The highytosolic redox state would stimulate the malate-aspartate shut-le that, through the entrance of malate in the mitochondria andormation of oxaloacetate, leads to an active transfer of reducedquivalents into the mitochondrial matrix. Thus, in the ADR group,ore reducing equivalents would be available in the mitochondria

nd the ETC complexes activity increase (Fig. 6). The ADR grouphowed a high reduction of MTT (Fig. 4A), suggesting high NADHehydrogenase (complex I) and/or succinate dehydrogenase (com-lex II) activities. The ADR group also presented a high reductionf TTC (Fig. 4B), indicative of high cytochrome c oxidase activ-ty (complex IV). When evaluating the subunits of cytochrome cxidase by MALDI-TOF-TOF, no significant differences were foundetween the treatment groups, indicating that the differences inhe metabolism were due to the its activity instead of alteredroteomics.

In ADR group, the lactate levels increased very slowly (Fig. 3B)nd alanine levels were even lower than control (Fig. 3C) suggestinghat, in the ADR group, the role of the mitochondria towards ATP for-

ation prevailed. Most certainly the pI shifts observed in ATP alphaynthase, a component of the F1 subunit, and cytochrome c (Table 2;

ig. 1) altered the efficacy of the mitochondrial coupled complexesTable 3). Altogether, these mitochondrial alterations may resultn increase mitochondrial membrane potential, reduced efficiencyf oxidative phosphorylation, and the formation of ROS (Lesnefsky

y 260 (2009) 84–96 93

et al., 2001). Accordingly, in a previous study, we shown that car-diomyocytes exposure to ADR, ADR with XXO, and XXO resulted inROS formation (Costa et al., 2009b). In the ADR group, the activityof ETC complexes increased as shown in TTC and MTT reductiontests and possibly led to higher mitochondrial ‘electron leakage’.To prevent more extensive oxidative damage, the cardiomyocytesin ADR group elicited an increase in manganese-containing mito-chondrial SOD (MnSOD) expression and in the total SOD activityto afford protection towards the increased ROS (Fig. 5). Presum-ably, the cardiomyocytes elicit protection mechanisms to survive,that are different whether the source of injury (namely ROS) resultsfrom the mitochondria or not.

On the other hand, the XXO group showed no differencesin TTC and MTT values to control, but maintained high levelsof glucose during all the incubation period. XXO group appar-ently preserved energy instead of actually producing it. In fact,as observed by the MALTI-TOF-TOF analysis, several spots relatedto ATP synthesis and carbohydrate metabolism had lower abun-dance in XXO, namely creatine kinase M chain, succinyl-CoA ligase,dihydrolipoyllysine-residue acetyltransferase component of PDHcomplex, and aconitase (Table 3).

Several cardiac redox regulatory proteins showed differences inthe treatments, namely peroxiredoxin-6 and SOD. Peroxiredoxin-6 abundance decreased in all treatment groups, especially in ADRwith XXO, presumably caused by oxidative stress-induced damage(Table 3). SOD abundance increased in the ADR group, as alreadymentioned and MnSOD increase in ADR group may have been acompensatory mechanism due to oxidative stress caused by theincreased mitochondrial ROS.

Stress proteins under altered biological conditions suffer rapidchanges (Snoeckx et al., 2001). Several stress response proteinsdecreased in the XXO group (Table 3), while, in a previous work,an early increase in inducible cytoplasmatic HSP70 was observed(Costa et al., 2009b) (spot referred as spot 42 in Table 3 is a fragmentand not the native protein). In the ADR with XXO group, besidesthe early increase in cytoplasmatic HSP70 (Costa et al., 2009b),another member of HSP70 family, 78 kDa glucose-regulated pro-tein precursor, was largely increased in the present work (Table 3).These new data suggests that the stress response in the XXO

exposed to adrenaline 500 �M (ADR), adrenaline 500 �M with xanthine and xan-thine oxidase (ADR with XXO), and XXO alone. Results are presented as means ± SDfrom 6 different experiments when compared to control (C). Statistical comparisonswere made using Kruskal–Wallis test, followed by the Student–Newman–Keuls posthoc test (*p < 0.05 vs. control; **p < 0.01 vs. control).

94 V.M. Costa et al. / Toxicology 260 (2009) 84–96

Fig. 6. Metabolic scheme demonstrating the main pathways of glucose utilization in the cardiomyocytes (not all intermediaries are shown for simplification). After glucoseuptake through the glucose transporter (GT) (1), it is activated and through glycolysis a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yieldpyruvate and NADH (2). Pyruvate can be converted into lactate by lactate dehydrogenase (LDH) (3) or to alanine by alanine aminotransferase (ALT) (4) in cytosol. Pyruvatemetabolism in the mitochondria requires its uptake by the pyruvate transporter (PT) followed by oxidation by pyruvate dehydrogenase (PDH) to acetyl-CoA (5). Acetyl-CoAenters the tricarboxylic acid (TCA) cycle where NADH and FADH2 are generated. These electron donors go into the electron transporter chain (ETC) (6) to form ATP andH2O. The cardiomyocytes were subjected to different treatments that elicited different responses. In the ADR group, the glucose transport and glycolytic pathways werestimulated and more reducing equivalents in the cytosol were generated. The ratio of alanine/lactate in the ADR was the lowest suggesting that reducing cytosolic equivalents(NADH/NAD+) were higher. This high NADH/NAD+ activated the malate-aspartate shuttle (MAS) that through the entrance of malate (MA) to the mitochondria and oxaloacetate(OX) formation leads to an active transfer of reduced equivalents to mitochondrial matrix from the cytosol (7). As more reducing equivalents would be transported in them re ‘elem in tha

Hi2

m(uetsicstm(rcX

rneips2a(t

itochondria, the ETC complexes activity increased (6) in the ADR group with moitochondria seems to be somewhat blocked favouring the pyruvate accumulation

nd less reducing equivalents (NADH) formation in the cytosol.

SP70 (spot 42), which can be correlated with the late increasen cytoplasmatic HSP70 observed in a previous work (Costa et al.,009b).

HSP90 is a HSP tightly associated with the proteasome thatediates the recognition of aberrant proteins for degradation

Whittier et al., 2004) and whose overexpression protects ATP reg-lating enzymes like PDH from oxidative injury (Veereshwarayyat al., 2006). In the present work, the HSP90 largely decreased inhe ADR with XXO, and XXO groups (Table 3), which can be respon-ible for the early inhibition of chymotrypsin-like activity observedn a previous work in those two groups (Costa et al., 2009b). It is notlear, however, if the HSP90 decrease was due to oxidative stress, asuggested by Beck et al. (2009) or if, instead, that decrease causedhe oxidative stress and the proteasome impairment, with accu-

ulation of altered structural proteins (Table 3) or quinoproteinsCosta et al., 2007). Furthermore, as HSP90 alters the activity of ATPegulating enzymes (Veereshwarayya et al., 2006), its decrease canontribute to the altered PDH abundance observed in the ADR withXO and XXO groups (Table 3).

In the present work, alterations in the abundance of severalegulatory proteins in the different treatments were observed,amely in cytochrome c [involved in the ETC and apoptosis (Costat al., 2009b)] and fatty acid-binding proteins. Prohibitin increasedn the ADR and ADR with XXO groups (Table 3). Mitochondrialrohibitin influences the assembly, stoichiometry, abundance or

tability of the oxidative phosphorylation complexes (Arrell et al.,006). Thus the alteration registered in the present work can revealltered mitochondrial activity in the ADR and ADR with XXO groupsTable 3). Serine/threonine protein phosphatase 2A, 65 kDa regula-ory subunit A, alpha isoform (spot 21) involved in the regulation

ctron leakage’. In the XXO group, on the other hand, the pyruvate oxidation in thee cytoplasm with higher formation of alanine and lactate, less glucose utilization,

of many cellular signalling molecules including metabolic enzymes,cell-surface receptors, cytosolic protein kinases, and transcriptionfactors and adenylate kinase isoenzyme 2 (spot 85) involved in ATPmetabolism, suffered a decrease in their abundance in the ADR andADR and XXO groups (Table 3).

Several other proteins identified in the present study showedaltered abundance, specifically VDAC-1. VDAC-1 is a highlyexpressed outer mitochondrial membrane channel that allowsthe flow of metabolites, including ATP and ADP. Recent studiessuggest that VDAC-1 is associated with the transport of crea-tine kinase and apoptogenic factors including cytochrome c viainteraction with its N-terminal domain (White et al., 2005). TheVDAC-1 decrease observed in XXO in the present work resultedfrom oxidative stress and may reveal a possible alteration in thatchannel, leading to the retention of cytochrome c in the mito-chondria, as previously observed (Costa et al., 2009b). Alterationsin VDAC-1 as a result of oxidative stress have been described inother studies (Taylor et al., 2003; Thiede et al., 2000). The increasein VDAC-1 in ADR group may contribute or be a result of alter-ation in ECT activity, as observed by the MTT and TTC reduction(Fig. 4).

In conclusion, the different treatments reveal dissimilar fea-tures. The ROS generating system (XXO) showed a particularnoxious effect in contractile proteins and metabolism that can becorrelated with myocardial stunning and altered mitochondrial

function caused by ROS at the onset of reperfusion. The ADR groupshowed increased activity in mitochondrial complexes, suggest-ing uncoupling of oxidative phosphorylation and/or increased ATPrequirements. Furthermore, ADR group had an increased expres-sion and activity of SOD, probably due to an adaptation to the

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ncreased mitochondrial ‘electron leakage’. The present work mayelp to disclose the mechanisms of I/R and HF, as both pathologiesre reported to have high ROS and catecholamine levels and showompromised heart function.

onflict of interest

None.

cknowledgements

This work received financial support from the Portuguese Statehrough “Fundacão para a Ciência e Tecnologia” (FCT) (projectPCDT/SAU-OBS/55849/2004). Vera M. Costa acknowledges FCT forer PhD grant (SFRD/BD/17677/2004).

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