J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 1 6 0 0 – 1 6 1 7

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Neuronal p60TRP expression modulates cardiac capacity

Manisha Mishraa, Arulmani Manavalana, Siu Kwan Szea, Klaus Heesea, b,⁎aSchool of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, SingaporebInstitute of Advanced Studies, Nanyang Technological University, 60 Nanyang View, Singapore 639673, Singapore

A R T I C L E I N F O

Abbreviations: p60TRP, p60 Transcription R⁎ Corresponding author at: Institute of Advance

Tel.: +65 6316 2848; fax: +65 6791 3856.E-mail address: klaus.heese@rub.de (K. H

1874-3919/$ – see front matter © 2011 Elseviedoi:10.1016/j.jprot.2011.11.034

A B S T R A C T

Article history:Received 19 October 2011Accepted 28 November 2011Available online 6 December 2011

Heart failure, including myocardial infarction, is the leading cause for death and theincidence of cardiovascular diseases is predicted to continue to rise worldwide. In thepresent study we investigated the whole heart proteome profile of transgenic p60-Transcription Regulator Protein (p60TRP) mice to gain an insight into the molecular eventscaused by the long-term effect of neural p60TRP over-expression on cardiac proteomechanges and its potential implication for cardiovascular functions. Using an iTRAQ (isobarictags for relative and absolute quantitation)-based proteomics research approach, we identi-fied 1148 proteins, out of which 116 were found to be significantly altered in the heart ofneural transgenic p60TRP mice. Based on the observed data, we conclude that in vivo neuralover-expression of transgenic p60TRP with its neuroprotective therapeutic potential signif-icantly affects cardiovascular capacities.

© 2011 Elsevier B.V. All rights reserved.

Keywords:p60TRPGpraspHeartMetabolism

1. Introduction

P60TRP is a member of the G-protein-coupled receptor associ-ated sorting protein (GPRASP) family. In recent years, exten-sive research has revealed numerous interacting partners ofG protein-coupled receptors (GPCRs) and one of which is theGPRASP family [1,2]. P60TRP is also known as GASP3 orBHLHB9 [3,4] and among the many distinguishing features ofp60TRP it is noteworthy that it contains a myc-type basichelix–loop–helix (bHLH) domain at its C-terminus which is aprotein structural motif that characterizes transcription fac-tors. Our studies have shown that p60TRP localized both inthe cytoplasm and the nucleus of cells and has been predom-inately expressed in the nervous system, the heart andskeletal muscle [3,4]. Recently, we have reported a newlyestablished transgenic p60TRP mice revealing its neuropro-tective mode of operation within the central nervous system(CNS) [4].

egulator Protein; iTRAQ,d Studies, Nanyang Techn

eese).

r B.V. All rights reserved

Recent advances in proteomics technologies offer opportu-nities to study the entire proteome of any sample in a singleexperiment. In comparison with previous gel-based method-ologies, the two-dimensional (2D) liquid chromatographycoupled with tandem mass spectrometry (2D-LC–MS/MS)-based multidimensional protein identification technology [5]combined with multiplex isobaric tag for relative and absolutequantification (iTRAQ) [6] provides an alternative approachfor quantitative proteomics profiling. This sensitive techniqueallows the simultaneous quantification of proteins in four-plex samples [7]. Recently, we have successfully applied thehigh-throughput iTRAQ-LC–MS/MS strategy in the area ofneuro-degenerative diseases [8,9]. Our p60TRP transgenicmice consistently revealed higher heart volumes comparedto wild-type littermates indicating a pivotal regulatory roleof p60TRP on neuromuscular junctions inmaintaining cardiacoutput. Accordingly, we applied the iTRAQ-based proteomicsto our clinically relevant transgenic mouse model for

isobaric tags for relative and absolute quantitation.ological University, 60 Nanyang View, Singapore 639673, Singapore.

.

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quantitative profiling of p60TRP-regulated cardiac genes. Sub-sequently, the iTRAQ-based proteomics–bioinformatics plat-form was used to generate the proteome comprising p60TRPregulated proteins from the transgenic mice-derived heart. Fi-nally, the altered expression of some of the regulated proteinswas validated by western blot analyses to delve into their car-diac activities (Fig. 1).

Since p60TRP is a novel proteinwithmanyunidentified fea-tures, our present investigations could further contribute to itsoperational assignment by providing substantial informationregarding its functional influence on neuromuscular junctionsand related myocardial diseases. Our in vivo results revealedthe long-term effect of neural p60TRP over-expression on

Fig. 1 – Schematic representation of the experimental design shoheart-derived tissue lysis, protein extracts were acetone precipitdigested. The quantitative proteomics analyses of transgenic p6multi-plex isobaric tags (114, 115, 116 and 117) for relative and abRepulsion–Hydrophilic Interaction Chromatography (ERLIC)-basetandem mass spectrometry (LC–MS/MS)-based multidimensionaanalyzed using ProteinPilot software and validated by quantitativinto various subgroups.

cardiac proteome changes and its potential implication forcardiovascular functions.

2. Materials and methods

2.1. Reagents

Unless indicated, all reagents used for biochemical methodswere purchased from Sigma-Aldrich (St. Louis, MO, USA). Ma-terials and reagents for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were from Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA). The iTRAQ reagent

wing biological and technical replicates. Followingated and quantified, ran in SDS-PAGE and subsequently0TRP mice-derived hearts was performed by labeling withsolute quantification (iTRAQ) reagent followed by Electrostaticd fractionation, and liquid chromatography coupled withl protein identification technology. The obtained data wase western blots. Finally, proteins were functionally classified

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multi-plex kit was bought commercially (Applied Biosystems,Foster City, CA, USA).

2.2. Antibodies

Anti-Atp5j2 (ATP synthase subunit f, 1:800, rabbit polyclonal;Abgent Inc., San Diego, CA, USA), anti-collagen-VI (Col6a3,1:1000, rabbit polyclonal; Abcam, Cambridge, UK), anti-Gapdh(Glyceraldehyde-3-phosphate dehydrogenase, 1:1000, mousemonoclonal; Abcam), anti-NAD(P) transhydrogenase (Nnt,1:800, monoclonal (8B4BB10); Abcam), and anti-peroxiredoxin5 (Prdx5, 1:800, polyclonal; Abcam).

2.3. Animal material

Experimental procedures, including the killing of animals,were in accordance with the International Guiding Principlesfor Animal Research (WHO) and approved by the local Institu-tional Animal Care & Use Committee (NTU-IACUC). Through-out the protocols, mice were provided with food and water adlibitum and maintained in controlled conditions (12 h light–dark cycle, 25 °C). All offspring were weaned onto standardchow at 21 days of age and metabolic tissue parameterswere assessed at 18 months of age. Mouse tissues (p60TRPtransgenic mice and wild-type littermates (C57BL/6J)) wereisolated after humane killing of the animals using approvedanesthetic methods. All efforts were made to minimize ani-mal suffering and to reduce the number of animals used[4,10].

2.4. Heart-tissue-specific protein expression analysis

Heart tissues were isolated from adult (~18 months) transgen-ic p60TRP mice and wild-type littermates (each group consist-ing of 3 males and 3 females for both batches BI and BII,respectively (Fig. 1)). Briefly, hearts were excised, snap-frozen in liquid nitrogen, and then powdered using a mortarand pestle. Upon the addition of lysis buffer (2% SDS, 0.5 MTriethyl ammonium bicarbonate buffer (TEAB), 1 Complete™protease inhibitor cocktail tablet (Roche, Mannheim, Germa-ny) and 1 PhosSTOP phosphatase inhibitor cocktail tablet(Roche)), the samples were vortexed for 1 min and incubatedon ice for additional 45 min prior to sonication (sonication pa-rameters: amplitude, 23%; pulse: 5 s/5 s for 5 min) using aVibra Cell high intensity ultrasonic processor (Jencon Scientif-ic Ltd, Leighton Buzzard, Bedfordshire, UK). After centrifuga-tion (20,000×g/4 °C/30 min), supernatants were collected andstored at −80 °C until further use. The protein concentrationwas quantified by a ‘2-D Quant’ kit (Amersham, Piscataway,NJ, USA) according to the manufacturer's protocol.

2.5. ITRAQ protocol [8,9]

2.5.1. Sample preparation - acetone precipitationSix hundred μg of total protein lysates wasmixed with six vol-umes of 100%-20 °C-chilled acetone, vortexed thoroughly andincubated overnight at −20 °C. The following day, sampleswere vortexed and centrifuged at 16,000×g for 30 min to pelletdown all proteins. The supernatant was discarded and thepellets were washed in 500 μl of 90%-20 °C-chilled acetone.

The washed pellets were allowed to air-dry at room tempera-ture (RT) for 15 min, dissolved in 100 μl of 200 mM TEAB and2% SDS a then incubated at 50 °C for 5–10 min with gentle ag-itation using a thermomixer (Eppendorf). The tubes were cen-trifuged at 16,000×g for 30 min. The supernatant wascollected and protein concentration was re-quantified usingthe ‘2-D Quant’ kit (Amersham).

2.5.2. SDS-PAGE and in-gel digestionTwo hundred μg of acetone-precipitated proteins was mixedwith loading dye denatured for 10 min in a thermo bath(Fine PCR, Seoul, Korea) and resolved up to 60% by SDS-PAGE. The gels were washed twice with autoclaved Milli-QWater (MQW) for 5 min each and fixed overnight on a SH30Lreciprocating shaker (Fine PCR) in 50% methanol and 10%Acetic Acid (AcOH). The gels were then washed with MQWthrice for 15 min each followed by in-gel digestion in a lami-nar flow hood (Gelman, Singapore). The gels were diced into1–2 mm pieces and transferred into tubes. 5 ml of 25 mMTEAB in 50% acetonitrile (ACN) buffer was added to the gelpieces, vortexed and left at RT for 10 min after which bufferwas discarded and the step repeated four times. Finally, 80%ACN in 20 mM TEAB was added, vortexed and the tubes wereleft at RT for 10 min. The supernatant was discarded and thesample tubes were left to air-dry for 30 min.

2.5.3. Reduction, alkylation, trypsin digestion and extractionStock solutions of 200 mM tris (2-carboxyethyl) phosphine(TCEP) in HPLC water (J.T. Baker, Mallinckrodt, Inc., Phillips-burg, NJ, USA) and 200 mM S-methyl methanethiosulfonate(MMTS) in isopropanol were prepared. 5 mM of TCEP in25 mMTEAB buffer was added to the dried gel pieces, vortexedand briefly spun before being incubated at 65 °C for 1 h toallow a reduction reaction to take place. Following this,10 mM MMTS in 25 mM TEAB buffer (tube was covered withaluminum foil) was added to gel pieces vortexed and brieflyspun to allow the alkylation reaction to proceed for 45 minat RT in dark. The supernatant was discarded. Gel pieceswere washed with 25 mM TEAB in 50% ACN buffer as de-scribed above and dehydrated by 100% ACN. Finally, thetubes were air-dried for 30 min. Digestion was performed intwo steps. First, 10 ml of 2.5 μg of trypsin in 25 mM TEAB buff-er was added to each sample and incubated at 4 °C for 15 minfor complete rehydration followed by the addition of further10 ml of 2.5 μg trypsin solution in a 37 °C incubator overnight.Subsequently, the tubes were spun briefly and the aqueousextract of the digested solution was collected. To the remain-ing gel pieces, 50% ACN and 1% AcOH was added, vortexedand incubated in a water bath sonicator for 30 min for the ex-traction of the peptides. The supernatant was transferred andcombined to the main sample tube. The extraction step wasrepeated 5 times. The trypsin digested peptides were pooledtogether and dried completely in the SpeedVac (Concentrator5301, Eppendorf) at 30 °C and stored at −20 °C.

2.5.4. Labeling of peptides with iTRAQ tags (4 plex)Each iTRAQ reagent tubes (tags— 114,115,116,117) had 70 μl of100% ethanol added and vortexed thoroughly. The dried pep-tides were dissolved in 30 μl of 500 mMTEAB (dissolution buff-er). Each iTRAQ tag was transferred to the respective peptide

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tubes and incubated at RT for 2 h with gentle agitation (ther-momixer). All individually labeled samples were then com-bined and dried in a SpeedVac at 30 °C.

2.5.5. DesaltingThe dried peptide samples were reconstituted in 500 μl of 0.1%formic acid (FA) and kept in the water bath sonicator for5 min. Fifty mg C18 cartridge (Sep-Pak® Vac C18 cartridges,Waters, Milford, MA) was conditioned thrice with 100% meth-anol pushed through at a rate of 2 to 3 drops per second via asyringe. The stationary phase was acidified three times with0.1% FA following the samemethod as conditioning. The sam-ple was loaded into the column and allowed to flow via grav-itational force and the flow-through was reloaded three times.Next, the sample loaded columnwas desalted twice with 0.1%FA and eluted using 75% ACN+0.1% FA. This C18 desaltingprotocol was performed thrice with the desalting wash solu-tion and the flow-through combined together. The sampleswere pooled together dried in SpeedVac and stored at −20 °C.

2.6. Electrostatic repulsion–hydrophilic interactionchromatography (ERLIC)

Eight hundred μg of iTRAQ-labeled peptides were fractionatedusing PolyWAX LP weak anion-exchange column (4.6×200mm,5 μm, 300 Å; PolyLC, Columbia, MD, USA), within the ShimadzuHPLC system (Kyoto, Japan). The HPLC gradient used composedof 100% solvent A (85% ACN, 0.1% AcOH, 10mM ammonium ac-etate, 1% FA, pH 3.5) for 5min, 0%–36% solvent B (30% ACN, 0.1%FA, pH 3.0) for 15 min, and 36%–100% solvent B for 25 min, and fi-nally 100%solvent B for 10 min, running for a total of 1 h at a flowrate of 1.0 ml min−1. A total of 29 fractions were collected. Thenumber of fractions was later reduced to 16 by pooling similarpeaks, as per the spectrum. The 16 sample tubes were dried inaSpeedVacandstored at−20 °C. Thedriedpeptides ineach sam-ple tube were reconstituted in 100 μl 0.1% FA for LC–MS/MSanalysis.

2.7. LC–MS/MS analysis

The samples were analyzed thrice (technical replicate=3) forLC–MS/MS using a Q-Star Elite mass spectrometer (AppliedBiosystems/MDS SCIEX) coupled with an online microflowHPLC system (Shimadzu). Multiple injections give a better cov-erage of the target proteome with superior statistical consis-tency. This is especially true for single peptide proteins asmore MS/MS spectral evidence was obtained frommultiple in-jections leading to higher confidence of peptide identificationand quantification [11]. The same pooled extracts were usedfor post-proteomics data validation using WB analysis. Thirtyμl of peptide mixture was injected and separated on a home-packed nanobored C18 column with a picofrit nanospray tip(75 μm i.d.×15 cm, 5 μm particles) (New Objectives, Wubrun,MA, USA) for each analysis. The samples were separated at aconstant flow rate of 30 μl/min with a splitter achieving an ef-fective flow rate of 0.3 μl/min. Data acquisition was performedin the positive ion mode, with a selected mass range of300–1600 m/z, and peptide ions with +2 to +4 charge stateswere subject to MS/MS. The three most abundant peptideions above 5 count threshold were selected for MS/MS and

each selected target ion was dynamically excluded for 30 swith 30 mDa mass tolerance. Automatic collision energy andautomatic MS/MS accumulation were used to activate smartinformation-dependent acquisition (IDA). With maximum ac-cumulation time being 2 s, the fragment intensity multiplierwas set to 20. The relative abundance of the proteins in thesamples was reflected by the peak areas of the iTRAQ reporterions.

2.8. Mass spectrometric data analysis

The data was acquired with the Analyst QS 2.0 software (Ap-plied Biosystems/MDS SCIEX). Using ProteinPilot Software 3.0,RevisionNumber: 114732 (Applied Biosystems), protein identifi-cation and quantification were performed. The peptides wereidentified by the Paragon algorithm in the ProteinPilot softwareand the differences between expressions of various isoformswere traced by Pro Group algorithm using isoform-specificquantification. The parameters used for database searchingwere defined as follows: (i) Sample Type: iTRAQ 4plex (PeptideLabeled); (ii) Cysteine alkylation: MMTS; (iii) Digestion: Trypsin;(iv) Instrument: QSTAR Elite ESI; (v) Special factors: None;(vi) Species: None; (vii) Specify Processing: Quantitate; (viii) IDFocus: biological modifications, amino acid substitutions;(ix) Database: concatenated ‘target’ (International ProteinIndex (IPI) mouse; version 3.55; 55,956 sequences) and ‘decoy’(the corresponding reverse sequences for false discovery rate(FDR) estimation); (x) Search effort: thorough. Pro Group algo-rithm was used to automatically select the peptide for iTRAQquantification, where the reporter peak area, error factor (EF)and p value were calculated. Auto bias-correction was carriedout on the acquired data to remove variations imparted as a re-sult of unequalmixing during the combination of thedifferentlylabeled samples. Tominimize the false positive identification ofproteins, a strict cutoff of unused ProtScore≥2 was used as thequalification criteria, which corresponds to a peptide confi-dence level of 99%. A FDR of 0.33% (<1.0%) was achieved. Thecutoff for up- or down-regulation (pre-defined at 1.2 and 0.83 re-spectively) was determined by using the p-value cut-off of 0.05to obtain the list of proteins with significant ratios. The p-value assigned by the ProteinPilot softwaremeasures the confi-dence of the real change in the protein expression level. Thendata analysis and functional classification were conductedusing online databases such as NCBI, UniProt, and Panther.

2.9. Post-proteomic data verification by SDS-PAGE andwestern blot analysis

Twenty μg of cell lysates was resolved by 8–12% SDS-PAGE at0.02 A of constant current and transferred to a polyvinylidinefluoride (PVDF) membrane (0.22 μm; Amersham) using the‘semi-dry’ transfer method (BioRad) for 60 min at 0.12 A inbuffer containing 25 mM Tris, 192 mM glycine, 20% methanol,and 0.01% (wt/vol) SDS. The membrane was blocked with 5%BSA (bovine serum albumin; BioRad) in phosphate-bufferedsaline (PBS) plus 0.1% Tween-20 (PBS-T) for 2 h at RT, washedthree times in PBS-T for 10 min each, and incubated with pri-mary antibody (diluted in 2% BSA in PBS-T) for overnight at4 °C. The membranes were washed as described above, incu-bated with HRP-conjugated secondary antibody for 1 h at RT,

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and developed using the ECL plus western blot detection re-agent (Amersham). X-ray films (Konica Minolta Inc., Tokyo,Japan) were exposed to the membranes before film develop-ment in a Kodak X-OMAT 2000 processor (Kodak, Ontario,Canada). For equal sample loading, protein quantificationwas performed using a ‘2D Quant’ kit (Amersham) with atleast two independent replicates. BSA was used as a standardfor protein quantification. To re-probe the same membranewith another primary antibody, Stripping solution (Pierce Bio-technology, Inc., Rockford, IL, USA) was used to strip themembranes. In addition, equal sample loading was confirmedusing Gapdh as a reference protein. Western blot experimentswere performed at least three times for statistical quantifica-tion and analyses (n=3), and representative blots are shown.Values (=relative protein expression) represent the ratio ofdensitometric scores (GS-800 Calibrated Densitometer andQuantity One quantification analysis software version 4.5.2;BioRad) for the respective western-blot products (mean±SD(standard deviation)) using the Gapdh bands as a referencefor loading control [12–15].

2.10. Statistical analysis

The data obtained in theWB analyses in this investigation areillustrated as mean±SD. Differences between the groups wereestablished using an unpaired Student's t-test while within-group comparisons were performed using the paired Stu-dent's t-test. SPSS (Statistical Products and Service Solutions)forWindows Version 19 was used to performANOVA followedby Fisher's Protected Least Significant Difference (PLSD) posthoc tests, when warranted. For the iTRAQ analysis ProteinPilotSoftware 3.0 was used. To be considered statistically signifi-cant, we required a probability value to be at least <0.05 (95%confidence limit, *p<0.05).

3. Results

3.1. Experimental design and identified proteins

All experiments were performed twice (batches BI and BII;Fig. 1) with each set repeated six times (six wild-type miceand six transgenic mice for BI and BII, respectively). We usedfour samples to perform iTRAQ (two wild-type (BI+BII) andtwo transgenic (BI+BII) samples) as six pooled biological repli-cates (all done twice: BI and BII). This was to ensure high con-fidence in the detection of cardiac proteins regulated byneural p60TRP. The quality of the dataset and instrumentalreproducibility was then confirmed by comparing and com-bining three technical replicates [8,9] after the samples werelabeled with 114, 115, 116 and 117 isobaric tags and processedin LC–MS/MS.

From the data obtained by two iTRAQ experiments(batches BI and BII), we identified a total of 1148 proteinswhich were further sorted based on a strict cutoff of unusedProtScore≥2 as the qualification criteria, that corresponds toa peptide confidence level of 99% and an applied FDR of0.33% (<1.0%), resulting in a total of 116 proteins that exhib-ited common trends in our experimental approach (Table 1).Essentially, 24 of these proteins were up-regulated (e.g.: Nnt,

Itih4/PK-120, Ppt1, Igk, Apoa1 and Col6a3 with p<0.05) andthe remaining 92 proteins were down-regulated (e.g.: Atp5j2,Idh3a, Ivd, Dpysl2, Hba-a2, Uqcrc1, D10Jhu81e, Slc25a11/12and Unc45 with p<0.05), with the cut-off for up- and down-regulation pre-defined at 1.2 and 0.83, respectively. Notably,proteins with possible cardio-protective roles, e.g. Nnt, Nlrx1,Txnl1, Tln2, Tmod1 Itih-4 and Col6a3, were up-regulated,while proteins involved in cell death e.g. Nduf, Atp5, Ckb/m,Vdac2/3, Prdx5, Unc45b and Ak1 were down-regulated.

3.2. Data verification by western blot, virtual 2D gelanalysis and bio-computational classification

In order to verify that the protein samples were indeed fromthe whole tissue proteome, the identified protein nameswere uploaded into JVirGel, a database software that createsa virtual 2D gel picture [16]. The proteins were categorizedbased on their isoelectric point and molecular weight (Fig. 2).The virtual 2D gel image showed that the samples collectedoriginated from the whole tissue proteome as the spots inthe gel were well dispersed.

We proceeded to use online databases (Panther, UniProt,and NCBI) to characterize the functions of these 116 proteins.During the classification process, our objective was also toidentify the proteins' sub-cellular localization and action(Fig. 3). It is of interest to note that a substantial proportionof the identified affected proteins are catalytic (~54%,Fig. 3A), regulating transporters (~30%, Fig. 3C) and are local-ized to the mitochondria (Fig. 3D).

Several of the other remaining proteins have importantroles in general transport mechanisms, including electrontransport, and other essential metabolic processes (Fig. 3Band Table 1). Many of these proteins are actually part ofmulti-protein complexes or are involved in various metabolicfunctions, suggesting that the regulation of a diverse array ofsignaling pathways is dependent upon expression of theseproteins.

3.3. Data validation by western blot

Following the database search and classification of proteins,western blots were performed on randomly selected proteinsto verify the iTRAQ values. Four proteins, Prdx5, Col6a, Nnt,and Atp5j2 were used for validation (Fig. 4). Gapdh was usedas an internal control to ensure equal loading of samples asits level was unchanged in the iTRAQ analysis.

Notably, the western blot images correlated very well andthus confirmed the iTRAQ values obtained.

3.4. Bio-computational network analyses

Functional partnerships between proteins constitute the core ofcomplex cellular phenotypes. The signaling networks formedby a protein with its interacting partners provide crucial scaf-folds for modeling to get insight into the mechanisms involvedin p60TRP-affected cardiac functions. STRING (Search Tool forthe Retrieval of Interacting Genes) is a database dedicated toprotein–protein interactions, including both physical andfunctional interactions. It weights and integrates informationfrom numerous resources, including experimental repositories,

Table 1 – Functional classification of differentially expressed heart proteins between wild type and transgenic p60TRP mice quantified by iTRAQ-based proteomics.

Protein ID Gene symbol Protein name Biological process No. of peptides(>95%) a

Tg/Wt heartiTRAQ ratio

p value

Circulation/lipid metabolismIPI00877236 Apoa1 Apolipoprotein A-I Lipoprotein metabolic process 6 2.40 0.02342IPI00775913 Apoa4 Apolipoprotein A-IV Lipoprotein metabolic process 2 1.27 0.43426

Acyltransferase activityIPI00153660 Dlat Dihydrolipoyllysine-residue acetyltransferase component

of pyruvate dehydrogenase complexCarbohydrate metabolic process 32 0.79 0.35017

IPI00154054 Acat1 Acetyl-CoA acetyltransferase Protein metabolic process 76 0.78 0.92978IPI00134809 Dlst Dihydrolipoyllysine-residue succinyltransferase component

of 2-oxoglutarate dehydrogenase complexCarbohydrate metabolic process 23 0.39 0.02328

Amino acid transmembrane transporter activityIPI00230754 Slc25a11 Mitochondrial 2-oxoglutarate/malate carrier protein Lipid metabolic process 38 0.47 0.00266IPI00320503 Slc25a42 Solute carrier family 25 member 42 Lipid metabolic process 1 0.60 0.66607IPI00308162 Slc25a12 Calcium-binding mitochondrial carrier protein Aralar1 Lipid metabolic process 45 0.58 0.00590

DNA binding/RNA helicase activityIPI00623951 Hist2h2ab Histone H2A type 2-B Nucleotide and nucleic acid metabolic process 10 0.58 0.38453IPI00117689 Ptrf Polymerase I and transcript release factor Nucleotide and nucleic acid metabolic process 24 0.79 0.55912IPI00120886 Ybx1 Nuclease-sensitive element-binding protein 1 Nucleotide and nucleic acid metabolic process,

mRNA processing2 0.24 0.67086

IPI00420577 Cand2 Cullin-associated NEDD8-dissociated protein 2 Nucleotide and nucleic acid metabolic process 6 0.53 0.05282IPI00230035 Ddx3x ATP-dependent RNA helicase DDX3X Nucleotide and nucleic acid metabolic process 2 0.14 0.06629IPI00409918 Eif4a2 Eukaryotic initiation factor 4A-II Nucleotide and nucleic acid metabolic process 2 0.19 0.29638

Kinase activityIPI00750256 Ak1 Adenylate kinase isoenzyme 1 Nucleotide and nucleic acid metabolic process 17 0.07 0.00116IPI00136703 Ckb Creatine kinase B-type Muscle contraction 25 0.16 0.51445IPI00127596 Ckm Creatine kinase M-type Muscle contraction 74 0.60 0.00412

Oxidoreductase activityIPI00129577 Aifm1 Apoptosis-inducing factor 1 Respiratory electron transport chain 74 0.46 0.13898IPI00132728 Cyc1 Cytochrome c1, heme protein Respiratory electron transport chain 40 0.32 0.03209IPI00117978 Cox4i1 Cytochrome c oxidase subunit 4 isoform 1 Respiratory electron transport chain 12 0.25 0.00370IPI00121309 Ndufs3 NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 Respiratory electron transport chain 22 0.83 0.0595IPI00129516 Uqcrh Cytochrome b-c1 complex subunit 6 Respiratory electron transport chain 3 0.19 0.37232IPI00874456 Dld Dihydrolipoyl dehydrogenase Respiratory electron transport chain 42 0.59 0.64707IPI00170093 Ndufs8 NADH dehydrogenase [ubiquinone] iron-sulfur protein 8,

mitochondrialRespiratory electron transport chain 8 0.53 0.09079

IPI00331332 Ndufa5 NADH dehydrogenase [ubiquinone] 1 alpha subcomplexsubunit 5

Respiratory electron transport chain 4 0.14 062819

IPI00153381 Uqcr10 Cytochrome b-c1 complex subunit 9 Respiratory electron transport chain 2 0.37 0.01968IPI00318750 Dhrs4 Dehydrogenase/reductase SDR family member 4 Lipid metabolic process 4 0.70 0.53916

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Table 1 (continued)

Protein ID Gene symbol Protein name Biological process No. of peptides(>95%) a

Tg/Wt heartiTRAQ ratio

p value

IPI00387430 Ndufb8 NADH dehydrogenase [ubiquinone] 1 beta subcomplexsubunit 8, mitochondrial

Respiratory electron transport chain 4 0.47 0.00084

IPI00387379 Decr1 2,4-dienoyl-CoA reductase, mitochondrial Lipid metabolic process 26 0.43 0.00966IPI00113196 Cox7a1 Cytochrome c oxidase polypeptide 7A1 Respiratory electron transport chain 4 0.20 0.13141IPI00323166 Nnt NAD(P) transhydrogenase Respiratory electron transport chain 5 5.39 0.00364IPI00131176 Mtco2 Cytochrome c oxidase subunit 2 Respiratory electron transport chain 47 0.53 0.21674IPI00120212 Ndufa9 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex

subunit 9Respiratory electron transport chain 46 0.36 0.00067

IPI00121288 Ndufb10 NADH dehydrogenase [ubiquinone] 1 beta subcomplexsubunit 10

Respiratory electron transport chain 10 0.36 0.04069

IPI00459725 Idh3a Isocitrate dehydrogenase [NAD] subunit alpha Tricarboxylic acid cycle 49 0.60 0.03371IPI00132531 Ndufb5 NADH dehydrogenase [ubiquinone] 1 beta subcomplex

subunit 5Nucleotide and nucleic acid metabolicprocess

6 0.41 0.07573

IPI00230715 Ndufa13 NADH dehydrogenase [ubiquinone] 1 alpha subcomplexsubunit 13

Electron transport chain 8 0.25 0.04349

IPI00915044 Prdx5 Peroxiredoxins-5 Thioredoxin superfamily, localizes tomitochondria and peroxisomes,peroxynitrite reductase, antioxidantand redox-signaling

21 0.19 0.00158

IPI00109169 Idh3g Isocitrate dehydrogenase [NAD] subunit gamma Tricarboxylic acid cycle 23 0.41 0.05269IPI00130804 Ech1 Delta(3,5)-delta(2,4)-dienoyl-CoA isomerase Lipid metabolic process 22 0.33 0.02433IPI00223757 Akr1b3 Aldose reductase Metabolic process 10 0.76 0.63884IPI00134961 Acadm Medium-chain specific acyl-CoA dehydrogenase Respiratory electron transport chain 61 0.64 0.81435IPI00471246 Ivd Isovaleryl-CoA dehydrogenase Respiratory electron transport chain 28 0.72 0.00193IPI00132042 Pdhb Pyruvate dehydrogenase E1 component subunit beta Lipid metabolic process 52 0.37 0.67781

Cytoskeletal protein bindingIPI00468140 Sorbs1 Sorbin and SH3 domain-containing protein 1 Cellular component morphogenesis 4 0.12 0.08846IPI00118153 Csrp3 Cysteine and glycine-rich protein 3 Cellular component morphogenesis 12 0.16 0.12265IPI00130102 Des Desmin Cellular component morphogenesis 86 0.77 0.24744IPI00113712 Tnnc1 Troponin C Muscle contraction 11 0.25 0.60447IPI00135182 Nrap Nebulin-related-anchoring protein Muscle contraction 2 1.94 0.19721IPI00125778 Tagln2 Transgelin-2 Muscle contraction 1 0.74 0.58705IPI00111265 Capza2 F-actin-capping protein subunit alpha-2 Cellular component morphogenesis 7 2.07 0.41167

Voltage-gated ion channel activityIPI00122547 Vdac2 Voltage-dependent anion-selective channel protein 2 Anion transport 20 0.35 0.02142IPI00876341 Vdac3 Voltage-dependent anion-selective channel protein 3 Anion transport 12 0.32 0.05736

Transaminase activityIPI00117312 Got2 Aspartate aminotransferase Cellular amino acid and derivative

metabolic process73 0.40 0.00166

IPI00228820 Gstm2 Glutathione S-transferase Mu 2 Immune system process 8 1.79 0.13774IPI00132002 Mgst3 Microsomal glutathione S-transferase 3 Protein metabolic process 2 0.70 0.54048IPI00225275 Pygm Glycogen phosphorylase Carbohydrate metabolic process 68 0.79 0.81974

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ChaperonesIPI00828649 Dnajc11 DnaJ homolog subfamily C member 11 Protein folding 1 0.62 0.44706IPI00788478 Unc45b Protein unc-45 homolog B Muscle organ development 2 0.44 0.00742IPI00129526 Hsp90b1 Heat shock protein 90 kDa beta member 1 Protein folding 4 0.37 0.18728ATP synthaseIPI00118986 Atp5o ATP synthase subunit O Coenzyme metabolic process 59 0.22 0.00219IPI00271986 Atp5j2 ATP synthase subunit f Respiratory electron transport chain 6 0.54 0.02255IPI00111770 Atp5k ATP synthase subunit e ATP synthesis coupled proton transport 7 0.25 0.11547IPI00876323 Atp5l ATP synthase subunit g ATP synthesis coupled proton transport 10 0.24 0.03961IPI00121550 Atp1b1 Sodium/potassium-transporting ATPase subunit beta-1 Cation transport 8 0.69 0.21059IPI00776084 Atp5c1 ATP synthase gamma chain ATP synthesis coupled proton transport 35 0.66 0.02611

Hydrolase activityIPI00114375 Dpysl2 Dihydropyrimidinase-related protein 2 Nucleotide and nucleic acid metabolic

process9 0.47 0.01737

IPI00310091 Ppp2r1a Serine/threonine-protein phosphatase 2A 65 kDaregulatory subunit A alpha isoform

Regulation of apoptosis 11 0.68 0.52507

IPI00315908 Mtp18 Mitochondrial 18 kDa protein Apoptosis 2 0.58 0.52109

Other proteinsIPI00453499 Iars2 Isoleucyl-tRNA synthetase Protein metabolic process 4 0.70 0.44136IPI00318671 Ehd4 EH domain-containing protein 4 Cell–cell signaling 7 2.70 0.48569IPI00380273 Gja1 Gap junction alpha-1 protein Signal transduction 1 0.84 0.08204IPI00228548 Eno3 Beta-enolase Carbohydrate metabolic process 81 0.65 0.36963IPI00129928 Fh Fumarate hydratase Nucleotide and nucleic acid metabolic process 52 0.50 0.01605IPI00119667 Eef1a2 Elongation factor 1-alpha 2 Signal transduction 32 0.57 0.42414IPI00403650 Prss2 Anionic trypsin-2 Protein metabolic process 17 0.37 0.05088IPI00122312 Fgg Fibrinogen gamma chain Immune system process 1 1.84 0.35935IPI00624663 A2m Alpha-2-macroglobulin 35 kDa subunit Signal transduction 7 2.78 0.06140IPI00128491 Aprt Adenine phosphoribosyltransferase Purine ribonucleoside salvage 1 0.12 0.12491IPI00132217 Fis1 Mitochondrial fission 1 protein Mitochondrial fission 4 3.31 0.29122IPI00850133 Kiaa0564 Uncharacterized protein KIAA0564 homolog ATP binding 12 0.55 0.07463IPI00221608 Samm50 Sorting and assembly machinery component 50 homolog Protein binding 7 0.33 0.16016IPI00128346 Cisd1 CDGSH iron sulfur domain-containing protein 1 Regulation of cellular respiration 4 0.34 0.38382IPI00229647 Tln2 Talin 2 Cell adhesion 4 3.10 0.44767IPI00605518 LOC100048867 Similar to Annexin A2 Positive regulation of binding 5 1.72 0.27980IPI00654054 Txnl1 Putative uncharacterized protein (Fragment) Glycerol ether metabolic process 1 1.50 0.04230IPI00845802 Hba-a2 Hemoglobin alpha, adult chain 2 Oxygen transport 56 1.75 0.00185IPI00857273 Clip1 161 kDa protein Zinc ion binding 1 1.22 0.60571IPI00655150 Tmod1 Tropomodulin 1 Myofibril assembly 9 3.31 0.52839

(continued on next page)

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Table 1 (continued)

Protein ID Gene symbol Protein name Biological process No. of peptides(>95%) a

Tg/Wt heartiTRAQ ratio

p value

IPI00312711 Itih4 PK-120 Hyaluronan metabolic process 1 8.39 0.04465IPI00462072 Eno1 Alpha-enolase Glycolysis 38 1.23 0.17643IPI00885793 Fga Fibrinogen, alpha polypeptide isoform 1 Signal transduction 5 1.22 0.07721IPI00648852 Ppt1 Palmitoyl-protein thioesterase 1 Regulation of phospholipase activity 2 2.65 0.01991IPI00918763 Uba52 Ubiquitin A-52 residue ribosomal protein fusion product 1 Ribosomal protein function 5 0.52 0.38042IPI00798592 Spna2 Spectrin alpha 2 Calcium ion binding 39 0.72 0.57468IPI00133977 Nap1l4 Putative uncharacterized protein Nucleosome assembly 1 0.68 0.78645IPI00468665 Myh13 Myosin, heavy polypeptide 13, skeletal muscle Actin binding 146 0.66 0.39713IPI00395100 Gm5409 Try10-like trypsinogen Proteolysis 11 0.58 0.02589IPI00648431 Myl4 Myosin, light polypeptide 4 Calcium ion binding 12 0.63 0.10180IPI00894588 Acadl Long-chain acyl-CoA dehydrogenase Fatty acid metabolism 69 0.48 0.00563IPI00653598 Uqcrc1 Ubiquinol-cytochrome c reductase core protein 1 Proteolysis 112 0.67 0.00145IPI00881401 Cpt2 Carnitine O-palmitoyltransferase Transferase 25 0.46 0.06867IPI00342603 Ogdhl Oxoglutarate dehydrogenase-like Glycolysis 26 0.57 0.05966IPI00169851 Ablim1 Putative uncharacterized protein, actin binding Cytoskeleton organization 3 0.32 0.30514IPI00267983 Acad12 Putative uncharacterized protein Acyl-CoA dehydrogenase activity 9 0.34 0.06460IPI00757372 Isoc2a Isochorismatase domain-containing protein 2A, mitochondrial Catalytic activity 4 0.65 0.76664IPI00133284 D10Jhu81e ES1 protein homolog Localizes to mitochondria 30 0.52 0.00364IPI00874570 Cp Ceruloplasmin Ferroxidase 1 1.24 0.67108IPI00889899 EG638833 Hypothetical protein Uncharacterized protein 14 1.26 0.12660IPI00830749 Col6a3 Col6a3 protein Collagen, key component of extracellular

matrices, microfibrillar protein68 1.82 0.02385

IPI00807999 Tmem173 Hypothetical protein LOC72512 isoform 1 Cytosolic DNA-sensing pathway 1 0.77 0.42049IPI00807819 LOC100044663 Similar to novel transmembrane domain

containing proteinUncharacterized protein 2 0.81 0.08467

IPI00850217 LOC100046119 Similar to ubiquitin-conjugating enzyme E2 UbcH-ben/ubiquitin-conjugating enzyme E2 N-like

Uncharacterized protein 1 0.78 0.30596

IPI00672367 Coq10a Coenzyme Q-binding protein COQ10 homolog A,mitochondrial

Uncharacterized protein 2 0.35 0.08385

IPI00849457 OTTMUSG00000020738 Similar to translocase of outer mitochondrialmembrane 20, Tomm20

Central component of the receptor complexresponsible for the recognition andtranslocation of cytosolically synthesizedmitochondrial preproteins. Together withTOM22 functions as the transit peptidereceptor at the surface of the mitochondrionouter membrane and facilitates the movementof preproteins into the TOM40 translocationpore (By similarity)

2 1.29 0.60423

IPI00404235 LOC100047867 Similar to mammary-derived growth inhibitor,Fabp3 - fatty acid binding protein 3, muscle and heart gene

FABP are thought to play a role in theintracellular transport of long-chain fattyacids and their acyl-CoA esters

25 0.78 0.23005

a The total number of peptides identified with >95% confidence.

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Fig. 3 – Pie chart depicting the identified proteins (characterizedcategory. Sub-cellular and functional categories were based on t(MGI) GO_Slim Chart Tool. Analyses were done considering varioC—Function. D—Sub-cellular localization.

Fig. 2 – Simulated 2D gel presentation of transgenic p60TRPmice-derived heart quantified proteins. The proteinsidentified by LC–MS/MS were uploaded onto JVirGel, anonline software used to create a virtual 2D gel image. Thisimage confirmed that the tissue-derived cell lysis performedwas adequate and the entire proteome within cells wasextracted. Isoelectric point and molecular weight valueswere generated from JVirGel at (http://www.jvirgel.de/).

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computational prediction methods and public text collections,acting as a meta-database that maps all interaction evidenceonto a common set of genomes and proteins [17]. Therefore,we used STRING to identify possible links among the 116 pro-teins obtained by p60TRP transgenic mice to enhance a bettercardiac systems-level understanding of cellular events in afunctional heart. Our current study reveals the functional linkamong proteins such as Nduf, Atp5, Vdac, Slc25a, Prdx5 andtheir potential connections to other metabolically modulatedproteins (Fig. 5).

Further bio-computational network analysis of the pro-teins identified in the transgenic p60TRP heart using the Inge-nuity Pathways Analysis (IPA) offered us additional valuableclues about the complex interactive link of proteins in theheart. In particular it might demonstrate the possible networkconnections among Atp1/5 proteins, various ion channels(Vdac2/3, Kcnj11 and Cacna1c) and other energy providingproteins (Ckb/m) as well as several other metabolic enzymessuch as AcadL/M and others (Fig. 6).

4. Discussion

GPCRs are 7-transmembrane-spanning (7TM) protein receptorsconsidered as the largest superfamily of membrane proteins

by iTRAQ) within the molecular function gene ontology (GO)he annotations of GO using the mouse genome informaticsus categories as indicated: A—Action. B—Process.

Fig. 4 – Western blot validation of iTRAQ results. Randomlyselected proteins significantly de-regulated in transgenic(Tg) p60TRP mice-derived hearts compared with controls(Wt, wild-type littermates). A—Nnt and Col6a levelsincreased in p60TRP transgenic mice while Prdx5 and Atp5j2levels were reduced. The western blots correlated to theiTRAQ values obtained. Gapdh was used as internal control.B—Quantitative analyses of the western blots shown in A.Western blot experiments were performed at least threetimes for statistical quantification and analyses (n=3).Values (=relative protein expression) represent the ratio ofdensitometric scores for the respective western blot productsand statistical error was indicated as mean±SD (*p<0.05)using the Gapdh bands as reference. C—The histogramindicates a similar close relationship between iTRAQ andwestern blot expression ratios. Transgenic p60TRPmice-derived hearts and control hearts iTRAQ expressionratios from selected proteins were consistent with thewestern blot results thus validating a strong agreement inthe expression data.

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that translate extracellular signals into intracellular messages.One of the ways in which their activity is controlled is by theprocess of desensitization and endocytosis. Following endocy-tosis, individual receptors can be sorted differentially betweenrecycling endosomes and lysosomes,which controls the revers-ibility of the silencing. Thus, endocytosis can either serve as amechanism for receptor resensitization by delivering receptorsback to the plasma membrane or facilitate receptor down-regulation by serving as the first step towards targeting the

Fig. 5 – STRING-9.0 analysis (Mus musculus at: (http://string-db.oDifferent line colors represent the types of evidence for the assocdirectly linked to the input as in Table 1) or white (nodes of a higconsist of up to eight lines: one color for each type of evidence.

receptors to lysosomes for degradation. The sorting of receptorsto the lysosomal pathway can be facilitated by interaction withan array of accessory proteins. One of these proteins is theGPCR-associated sorting protein 1 (GASP-1 or GPRASP1), whichspecifically targets several 7TM-GPCRs to the lysosomal path-way after endocytosis [2,18]. P60TRP was recently found as anovel member of the GPRASP family [3] and in vivo relevanceof p60TRP-dependent signaling has been verified in a mousemodel over-expressing neural p60TRP [4]. Here, weinvestigated the long-term effect of neural p60TRP over-expression on the cardiac proteome and identified altered ex-pression of 116 proteins. Successively, 14 proteins with alteredfunctions in cardiac diseases were selected from the list of 116proteins: 7 up-regulated (Nnt, Col6a3, Txnl1, Nlrx1, Tln2,Tmod1 and Itih-4) and 7 down-regulated (Nduf, Atp5, Ckb/m,Vdac2/3, Prdx5, Unc45b and Ak1). In the following sections, thespecific functional roles of these proteins, their relevance to car-diac diseases and the potential consequences of their modifiedexpressions are discussed.

4.1. Neuronal p60TRP expression mediates theup-regulation of proteins crucially involved in cardiac functions

4.1.1. NntMitochondrial (mt) NAD(P) transhydrogenase is an integral pro-tein of the innermt-membrane that is encoded by theNnt gene.In mice, the Nnt expression is highest in heart tissue and pro-tects against oxidative stress [19,20]. The enzyme coupleshydride transfer between NAD(H) and NADP(+) to proton trans-location across the innermt-membrane.Undermost physiolog-ical conditions, the enzyme uses energy from the mt-protongradient to produce high concentrations of NADPH. TheresultingNADPH is used for biosynthesis and free radical detox-ification [21]. Thus, the physiological role of the proton-translocating transhydrogenase has generally been assumedto generate mt-NADPH. Mt-NADPH can be used in a number ofimportant reactions/processes, e.g., biosynthesis, maintenanceof GSH, apoptosis, aging etc. NADPH is also needed for the scav-enging ofROS suchas superoxide anion, hydrogenperoxide, hy-droxy radical generated as ‘by-products’ of certain physiologicalcellular processes: e.g. oxidative phosphorylation and activitiesof monoamine oxidase (Mao), tyrosine hydroxylase (Th), nitricoxide synthase (Nos), cyclooxygenase (Cox) or lipoxigenase(Lox).

Cardiac ATP-sensitive potassium (KATP) channels are ionchannels which are critical for stress adaptation in heart[22,23]; the novel cGMP/PKG/ROS/calmodulin/CaMKII signal-ing pathway regulates cardiomyocyte excitability by openingKATP channels and contributes to cardiac protection againstischemia injury [24]. Accordingly, Nnt malfunction may dis-turb mt-metabolism leading to less ATP production, and con-sequently enhanced KATP channel activity [25]. Thus, inp60TRP transgenic mice enhanced Nnt might cause elevated

rg/)) of transgenic p60TRP-modulated proteins in the heart.iation. Network display: nodes are either colored (if they areher iteration/depth). Edges, i.e. predicted functional links,

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ATP production and reduced KATP channel activity which is inagreement with reduced Ak1 expression. Besides, p60TRPmay protect heart from potential ischemia-related injuriesby activating the PI3 kinase-Akt pathway and mt-KATP chan-nel activities [4,22]. Therefore, in p60TRP transgenic mice theenhanced Nnt gene activity makes the heart eventually lesssensitive for oxidative stress and stabilizes it for ischemia in-jury [20].

4.1.2. Col6a3Collagen, the most abundant protein in animals, is a key com-ponent of extracellular matrices. Collagens not only provideessential structural support for connective tissues, but theyare also intimately involved in controlling a spectrum of cellu-lar functions such as growth, differentiation, andmorphogen-esis. Collagen VI (Col6) is a microfibrillar protein found invirtually all connective tissues. It is composed of three distinctsubunits, alpha1(VI), alpha2(VI), and alpha3(VI), which associ-ate intracellularly to form triple helicalheterotrimeric monomers, then dimers and tetramers. Thesecreted tetramers associate end-to-end to form beaded mi-crofibrils [26]. Col6 has anti-apoptotic functions mediated viathe PI3K/Akt pathway [27] and mutations in COL6A3 cause se-vere and mild phenotypes of Bethlem myopathy and Ullrichcongenital muscular dystrophy [28,29]. Increased levels ofthe non-fibrillar Col6 may also reflect an early protectiveremodeling system in patients with acute myocardial infarc-tion [30–33]. In this respect it is worth to note that the angio-tensin (Ang)/GPCR-Mas receptor (Ang(1–7)/MasR) axis plays akey role in the maintenance of the collagen structure andfunction of the heart [34]. Ang(1–7)/MasR signaling is associat-ed with protective cardiac responses. Co-localization of MasRand the small GTPase Rab5 indicates that the MasR is inter-nalized through a clathrin-mediated pathway and targetedto early endosomes after Ang-(1–7) stimulation [35].

4.1.3. Txnl1Thioredoxin-related Protein 32 Txnl1/TRP32 is an arsenite-regulated thiol reductase of the proteasome 19 S particle anda redox-active cofactor of the 26 S proteasome. Txnl1 is oneexample of a direct connection between protein reductionand proteolysis, two major intracellular protein quality con-trol mechanisms [36,37]. In addition, Txnl1 plays a regulatoryrole in fluid phase endocytosis whereby Txnl1 acts as an effec-tor of oxidants or a redox sensor by converting redox changesinto changes of GDI capacity to capture the small GTPaseRab5, which in turn modulates fluid phase endocytosis [38].The small GTPase Rab5 is a crucial regulatory component ofthe endocytic pathway. Activation of Rab5 is mediated byGDP–GTP exchange factors (GEFs) that generate the Rab5-GTP complex. In addition, Rab5 regulates the endocytic recy-cling of Kcnq1/Kcne1 potassium channels. Thus, as a GPRASPfamily member p60TRP may modulate those mechanisms byinterfering with the Txnl1/Rab5 and MasR/Rab5 pathways[35,39–42].

4.1.4. Nlrx1An elevated cardiac Nlrx1 protein level in p60TRP transgenicmice further confirms its protective action [43]. Nlrx1 is amt-outer-membrane Nod-like receptor (Nlr) that amplifies

NF-kappaB and Jnk-dependent signaling pathways by induc-ing ROS production [44].

4.1.5. Tln2Talins are large adaptor proteins that link the integrin familyof adhesion molecules to F-actin. In vertebrates, there aretwo talin genes. Tln1 is essential for integrin-mediated celladhesion; the role of Tln2 is largely unclear. Recently it hasbeen shown that Tln2 is a large and complex gene encodingmultiple transcripts and protein isoforms [45]. Tln1 and Tln2connect integrins to the actin cytoskeleton and regulate theaffinity of integrins for ligands. Tln1 and Tln2 are requiredfor myoblast fusion, sarcomere assembly and the mainte-nance of myotendinous junctions [46]. The vinculin–talin–integrin system has a role in the transduction of mechanicalforce to the extracellular matrix. It is also attributed a keyrole to the regulation of action potential duration in cardiacmyocytes [47]. An up-regulation of Tln2 protein levels intransgenic p60TRP mice could eventually have supportiveconsequences by strengthening the cardiac muscleactivities.

4.1.6. Tmod1Tmod1 is a multifunctional protein: its actin filament cappingactivity prevents thin filament elongation, whereas its inter-action with tropomyosin promotes thin filament polymeriza-tion [48]. Actin (thin) filament length regulation and stabilityare essential for striated muscle function and any disruptionin the Tmod1 gene expression or Tmod1 protein levels maycompromise cardiomyocyte development in murine embry-onic stem cells by arrestingmyofibril maturation thus causingheart failure [49].

4.1.7. Itih-4Itih-4 is expressed as a strong 3.1-kb transcript in liver and toa lesser extent in lung and heart tissue [50]. Itih-4 encodes a942 amino acid protein containing two EF-hand (helix–loop–helix) motifs with a unique short loop, with a potentialCa2+-binding function. It may thus play a pivotal Ca2+-de-pendent role in heart action and regenerative cardiac pro-cesses [51].

4.2. Neuronal p60TRP expression mediates thedown-regulation of proteins crucially involvedin cardiac function

4.2.1. NdufRecently, NADH dehydrogenase (ubiquinone) (NDUFS3) dereg-ulation has been associated with heart failure in human [52].Another case report showed that mutations in mt-Ndufgenes might be responsible for the development of Leigh'sdisease [53], a rare neuro-metabolic disorder that affects theCNS. In Leigh's disease, crucial cells in the brain stem havemutated mtDNA, creating poorly functioning mitochondriawhich causes chronic lack of energy in these cells, which, inturn, affects the CNS and inhibits motor functions. Basically,the disease is characterized by movement disorders includingdystonia, rigidity, tremor, chorea, hypokinesia, myoclonus,and tics [54]. However, the significance of cardiac Nduf ex-pression changes in transgenic neural p60TRP mice awaits

Fig. 6 – Network analysis of proteins identified in the transgenicp60TRP heart using the IPA. Eightmergedmajor networkswereanalyzed based on the iTRAQ data of proteins expressed inheart: (i) 15 (underlined) human genes, identified by our iTRAQanalysis (ACADM, ACAT1, ATP1A1, ATP1B1, ATP5C1, ATP5O,CKM,DLAT,DLD,DLST, ENO1, ENO3,GOT2,KCNJ11, PLN, PYGM,UQCRC1, VDAC2), are involved in various genetic disorders,neurological diseases, as well as in skeletal and muscular dis-orders. Network (ii) disclosed two (underlined) genes (ACADL,KCNJ11, PDHB) of our experimental iTRAQ analysis that havebeen shown to be involved in various genetic disorders,metabolic diseases, and lipid metabolism disorders. Network(iii) revealed one (underlined) gene (CP, TNF) involved in dis-eases related to molecular transport, genetic disorders, andophthalmic diseases. Network (iv) unraveled the genes (CSRP3,GLRX3) that are involved in cardiovascular systemdevelopmentand function, organmorphology, and cardiac hypertrophy.Network (v) presented two genes (FGG, NFkB (complex)) that areinvolved in genetic disorders, hematological diseases, andcellular assembly and organization. Network (vi) depicted thosegenes (GIMAP5, HSP90B1) that are involved in cellulardevelopment, skeletal and muscular system development andfunction, and tissue morphology. Network (vii) demonstratedgenes (e.g. CACNA1C) that are involved in cell morphology,cellular function andmaintenance, and drug metabolism. Andfinally network (viii) highlighted the genes (APOA1, CAV1) thatare involved in cardiovascular diseases, lipid metabolism, andsmall molecule biochemistry. The solid lines refer to a directprotein–protein interaction, while dotted lines show an indirectrelationship among the iTRAQ-based identified genes.

Fig. 7 – Neuronal over-expression of p60TRP affectsvarious cardiac protein functions. Red color: activation ofNnt-, Txnl1/Rab5-, Ang/MasR/Rab5/Col6a3-signalingpathways; green color: inhibition of Ckb/m-, Atp5- andAk1-signaling pathways; which in turn modulate pivotalcardiac myocytes' K+ channels' activities and cardiac output.

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further detailed investigations with a closer focus on cardiacneuromuscular junctions.

4.2.2. Atp5Mt-ATP synthase catalyzes ATP synthesis, utilizing an electro-chemical gradient of protons across the inner membrane

during oxidative phosphorylation. It seems obvious thateven intermittent and minor impairment of this highly im-portant enzyme could deprive the heart tissues of energy atcrucial times, which may predispose or contribute to cardiacdiseases [55].

4.2.3. Ckb/mCreatine kinase-B/M type–Increased fetal cardiac Ckm and Atpsynthase may contribute to acceleration of glycolysis in thepreterm heart [56]. Besides, levels of Ckb with an M-type sub-unit are significantly higher in non-survivor patients whencompared with survivors with acute myocardial infarction(AMI) [57]. Interestingly, p60TRP reduced the level of Ckbsupporting its protective nature. Furthermore, similar toNDUFS3, CKM has also been shown to be down-regulated inpatients with heart failure [52].

4.2.4. Vdac2/3Voltage-dependent anion channels (Vdac) are pore formingproteins predominantly found in the outer mt-membraneand are thought to transport Ca2+. Vdac2 modulates restingCa2+ sparks, but not action potential-induced Ca2+ signalingin cardiac myocytes [58]. P60TRP-mediated decreased Vdac2/3 levels may also be part of a protective cardiac remodeling

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system to improve cardiac functions as previously shown fol-lowing myocardial infarction [59].

4.2.5. Prdx5Peroxiredoxins (Prdxs), the antioxidant components of the thior-edoxin superfamily, is a family of antioxidant and redox-signaling proteins. There are currently six known Prdx enzymesthat protect cells and tissues from damage caused by ROS inmammals [60]. Prdx5 itself is a peroxynitrite reductase [61] thatis expressed ubiquitously and localizes to mitochondria andperoxisomes [62] and possesses antioxidant activity equivalentto that of catalase [63]. Prdx5 plays a protective role against

Fig. 8 – Cardiovascular network analysis of iTRAQ-based proteomanalysis for the understanding of how the identified proteins wocardiovascular diseases and heart failure-related metabolic signcardiovascular system induced by neural expression of p60TRP.

oxidative stress and is essential for protection against apoptosis[64–68].

Prdxs are plentiful within the heart; however, their cardiacfunctions are poorly understood. Cardiac Prdxs undergo acomplex series of redox-dependent structural changes in theheart in response to cardiac oxidant challenge with its sub-strate hydrogen peroxide [69]. Similar to NDUFS3 and CKM,PRDX2 has also been shown to be down-regulated in patientswith heart failure [52]. Prdxs have also gained recognition asimportant redox regulating molecules relevant to the mecha-nisms underlying ischemia–reperfusion injury [70]. Thus,elevated neural levels of p60TRP may reduce stress-related

ic metabolism in transgenic p60TRP heart using IPA. IPArk together by protein–protein interactions in the context ofaling pathways that affect cellular changes in the

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cardiovascular signaling pathways and could induce protec-tive cardiac mechanisms that require less Prdx5 activity.

4.2.6. Unc45bMyosin folding and assembly in striatedmuscles ismediated bythe general chaperones Hsc70 and Hsp90 and amyosin specificco-chaperone, Unc45. Two Unc45 genes are found in verte-brates, including a striated muscle specific form, Unc45bwhich is expressed in both skeletal and cardiac muscles [71].Mammalian Unc45b is a cytosolic protein that forms a stablecomplex with Hsp90, selectively binds the unfolded conforma-tion of themyosinmotor domain, and promotesmotor domainfolding [72]. Recent studies indicate that the expression levels ofUnc45b must be precisely regulated to ensure normal myofibrilorganization. The Unc45 chaperone is critical for establishingmyosin-basedmyofibrillar organization and cardiac contractili-ty in the drosophila heartmodel [73]. Loss or over-expression ofUnc45b leads to defective myofibril organization [71].

4.2.7. Ak1 isoform 2 of adenylate kinase isoenzyme 1Detailed expression analyses about AK isozymes in humanheart and skeletal muscle revealed that the AK2 protein wasfound only in heart, whereas AK1 was detected in both tissues.These tissue-specific expressions of the AK isozymes in humanmight suggest the presence of an organ-specific regulation ofthe AK1 and AK2 genes including a post-transcriptional control[74]. Transduction of energetic signals intomembrane electricalevents governs vital cellular functions. Central to the regulationof such diverse cellular processes are the metabolism sensingK+

ATP channels. Deletion of the Ak gene compromised nucleo-tide exchange at the channel site and impeded communicationbetween mitochondria and nucleotide-gated K+

ATP channels,rendering cellular metabolic sensing defective. Assigning asignal-processing role to Ak identifies a phospho-relay mecha-nism essential for efficient coupling of cellular energetics withK+

ATP channels and associated functions [75]. Matching bloodflow to myocardial energy demand is vital for heart perfor-mance and the molecular mechanisms responsible fortransduction ofmyocardial energetic signals into reactive vaso-dilatation are still hardly described. Ak, associated with AMPsignaling, is a sensitive reporter of the cellular energy state,yet the contribution of this phosphotransfer system in couplingmyocardial metabolism with coronary flow has not been ex-plored in detail. A recent study with Ak1 knockout mice dis-rupted the synchrony between inorganic phosphate P(i)turnover at ATP-consuming sites and gamma-ATP exchangeat ATP synthesis sites. Ak1 gene deletion blunted vascularAk phosphotransfer, compromised the contractility–coronaryflow relationship, and precipitated inadequate coronary reflowfollowing ischemia–reperfusion. Deficit in Ak activity abrogatedAMP signal generation and reduced the vascular Ak/Ck activityratio essential for the response of metabolic sensors. Thesarcolemma-associated splice variant Ak1beta facilitated aden-osine production, a function lost in the absence of Ak activity.Ak1 phosphotransfer thus transduces stress signals into ade-quate vascular response, providing a linkage between cell bio-energetics and coronary flow [76]. Ak1 protein expressionlevels are thus crucial for the cardiovascular capacity and ap60TRP-mediated down-regulation could cause severe negativeside effects.

Thus, neuronal over-expression of p60TRP affects variouscardiac protein functions which in turn modulate cardiacmyocytes' K+ channels' activities that are crucial for the con-trol of cardiac output (Fig. 7).

5. Conclusions

Our data has shed new insights on the possible involvementof the novel neuroprotective protein p60TRP in cardiovascularfunctions suggesting a connection between these two organs;brain and heart. Though neuronal over-expression of p60TRPmay contribute to improved neural and cognitive activitiesin the brain [4], in the long term it may also affect cardiovas-cular capacities (Fig. 8) probably by regulating neuromuscularjunctions. However, further functional in/ex vivo studies arerequired to unravel the functional significance of p60TRPand its direct effect on cardiovascular performance and at car-diac neuromuscular junctions.

Acknowledgments

This study was supported by the Institute of Advanced Stud-ies, Nanyang Technological University (NTU) and by grantsfrom NTU (SBS/SUG/22/04) and from the Agency for Science,Technology and Research (A*STAR) (BMRC/04/1/22/19/360) toKH.

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