Proteomic Profiling of H-Ras-G12V Induced ... - ACS Publications

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Proteomic Profiling of H-Ras-G12V Induced Hypertrophic Cardiomyopathy in Transgenic Mice Using Comparative LC-MS Analysis of Thin Fresh-Frozen Tissue Sections Bih-Rong Wei,† R. Mark Simpson,† Donald J. Johann, Jr.,‡ Jennifer E. Dwyer,† DaRue A. Prieto,§ Mia Kumar,† Xiaoying Ye,§ Brian Luke,∥ Heather R. Shive,† Joshua D. Webster,† Shelley B. Hoover,† Timothy D. Veenstra,§ and Josip Blonder*,§ †

Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, United States ‡ Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, United States § Laboratory of Proteomics and Analytical Technology, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702, United States ∥ Advanced Biomedical Computing Center, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702, United States S Supporting Information *

ABSTRACT: Determination of disease-relevant proteomic profiles from limited tissue specimens, such as pathological biopsies and tissues from small model organisms, remains an analytical challenge and a much needed clinical goal. In this study, a transgenic mouse disease model of cardiac-specific H-Ras-G12V induced hypertrophic cardiomyopathy provided a system to explore the potential of using mass spectrometry (MS)-based proteomics to obtain a disease-relevant molecular profile from amount-limited specimens that are routinely used in pathological diagnosis. Our method employs a twostage methanol-assisted solubilization to digest lysates prepared from 8-μm-thick fresh-frozen histological tissue sections of diseased/experimental and normal/control hearts. Coupling this approach with a nanoflow reversed-phase liquid chromatography (LC) and a hybrid linear ion trap/Fourier transform-ion cyclotron resonance MS resulted in the identification of 704 and 752 proteins in hypertrophic and wild-type (control) myocardium, respectively. The disease driving H-Ras protein along with vimentin were unambiguously identified by LC-MS in hypertrophic myocardium and cross-validated by immunohistochemistry and western blotting. The pathway analysis involving proteins identified by MS showed strong association of proteomic data with cardiovascular disease. More importantly, the MS identification and subsequent crossvalidation of Wnt3a and β-catenin, in conjunction with IHC identification of phosphorylated GSK-3β and nuclear localization of β-catenin, provided evidence of Wnt/β-catenin canonical pathway activation secondary to Ras activation in the course of pathogenic myocardial hypertrophic transformation. Our method yields results indicating that the described proteomic approach permits molecular discovery and assessment of differentially expressed proteins regulating H-Ras induced hypertrophic cardiomyopathy. Selected proteins and pathways can be further investigated using immunohistochemical techniques applied to serial tissue sections of similar or different origin. KEYWORDS: H-Ras, hypertrophic cardiomyopathy, fresh frozen tissue, methanol-based solubilization/digestion, comparative proteomic profiling, pathway analysis, method development

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INTRODUCTION Mass spectrometry (MS) is striving to become a common means of analyzing tissue specimens for the development of disease-relevant biomarker candidates. Required is the continued optimization and development of methods capable of discriminating diseased/experimental from healthy/control tissue specimens that would facilitate the molecular characterization of tissue phenotypes directly from in vivo sources.1 While many MS-based proteomic approaches have been developed and routinely used for molecular profiling of cultured cells, these in vitro proteomes do not accurately resemble those in vivo.2 Therefore, the ability to directly profile © 2012 American Chemical Society

tissues as well as other types of clinical specimens in vivo is critical. To date, efficient proteome-wide profiling of histopathological clinical specimens remains challenging. This is primarily due to the high complexity of protein and cellular components in tissue specimens, specific requirements for upstream LC-MS sample preparation, and their limited supply.3 Proteomic profiling of tissues needs to provide in-depth characterization of molecular changes that exist in vivo, permitting elucidation of tangible signaling pathways and Received: June 27, 2011 Published: January 4, 2012 1561

dx.doi.org/10.1021/pr200612y | J. Proteome Res. 2012, 11, 1561−1570

Journal of Proteome Research

Article

replacing the doxycycline containing diet with a regular diet for 4 weeks, a period sufficient for approximately 80% of M/R mice to develop cardiac lesions.8 The study was performed under review by the institutional animal care and use committee. Diseased as well as healthy/control heart ventricles were embedded in cryostat mounting medium (TISSUE-Tek O.C.T., Sakura Finetek, Inc., Torrance, CA) using a standard histopathological protocol, and portions were also snap frozen for subsequent biochemical analysis. The procured specimens were stored at −80 °C until sectioning. Prior to LC-MS analysis, the presence of cardiac hypertrophy was validated by measuring the heart (H) to body (B) weight (W) ratio along with the analysis of hematoxylin and eosin (H&E) stained myocardial tissue slices. Ventricle tissue slices were obtained from diseased M/R mice with HW/BW ratios of 16.3 mg/g (lesion score 2) and 16.8 mg/g (lesion score 3), while HW/BW ratios of healthy/control animals were 9.4 mg/g and 8.4 mg/g (both lacking myocardial lesions; lesion score 0). H&E stained slides were reviewed by a certified pathologist to confirm disease phenotype.13 A lesion score (0−3) was assigned to each specimen, as previously described.8 Additional specimens of ventricle myocardium were preserved by immersion in aldehyde fixation buffer and processed routinely as paraffinembedded tissue specimens for sectioning and mounting on microscope slides. These latter specimens provided additional material for immunohistochemistry (see below). For proteomic analysis, two diseased and two control heart ventricles (biological replicates) were included for study, and from each, two thin tissue slices (technical replicates) were processed and analyzed by LC-MS. After sectioning, each 8μm-thick unstained tissue slice was mounted on a glass microscope slide (HistoServ, Inc., Germantown, MD). Individual frozen tissue slices were scraped off of the glass slide using a razor blade and transferred into a 1.5-mL microcentrifuge tube containing 80 μL of 12.5 mM ammonium bicarbonate (pH 7.5) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Tissue lysis and homogenization was carried out by incubating each sample on ice for 30 min followed by sonication in a water bath for 10 min. After boiling in a water bath for 10 min, samples were then cooled to room temperature and protein concentration was determined using the BCA assay. Equal quantities of proteins were reduced for 30 min at room temperature using tris(2-carboxyethyl)phosphine (TCEP) (1 mM final concentration). After alkylation, employing 3 mM iodoacetamide (final concentration) for 30 min at room temperature, samples were lyophilized to dryness. Each sample was then resolubilized in 20% methanol/50 mM ammonium bicarbonate (v/v) buffer (pH 7.9) and digested at 37 °C for 3 h using trypsin at a 1: 20 trypsin/protein ratio and subsequently lyophilized to dryness.14 Lyophilized digestate was resuspended in 60% methanol/50 mM ammonium bicarbonate (v/v) buffer (pH 7.9) and digested again at 37 °C overnight using trypsin at a 1:20 trypsin/protein ratio. After centrifugation at 20,000g for 15 min, the supernatant was lyophilized to dryness and resuspended in 0.1% trifluoroacetic acid (TFA) prior to cleaning with a ZipTip C18 tip column (Millipore, Billerica, MA) in accordance with the manufacturer’s instructions. Sufficient material was derived from each section for two independent MS analyzer runs.

detection of multiple protein alterations responsible for the pathology under study.4 A proteome-wide characterization of tissues derived from in vivo sources should allow better elucidation of protein species as biomarker candidates and/or potential drug targets.5 Extensive reviews on tissue proteomics have been published elsewhere.1,6,7 The present study describes a proteomic method for molecular profiling of thin fresh-frozen tissue sections routinely used in a pathology diagnostic workflow. A two-stage methanol-assisted solubilization/digestion, optimized for preparation of thin myocardial tissue sections was coupled with high-resolution and high accuracy LC-MS analysis. Myocardial specimens were obtained from a previously described and validated transgenic mouse model of a pathogenic myocardial hypertrophy induced by targeted expression of the activated mutant H-Ras-G12V in the myocardium.8 It is known that Rasdependent pathway activation plays a significant role in cardiac hypertrophy resulting from stimuli, such as pressure overload, epinephrine, and endothelin-1.9−12 Therefore, the inducible genetic expression of activated H-Ras provides a means to study pathogenic myocardial hypertrophy while minimizing the background genetic and interindividual variability that is often encountered in human subjects. Subtractive proteomic analysis coupled with differential spectral count based quantitation led to the identification of cardiac disease-relevant proteins that were differentially expressed in diseased vs healthy hearts. Cross-validation of LC-MS data using immunohistochemistry (IHC) and western blot (WB) analyses documented the presence and activation of Wnt/β-catenin signaling events in H-Ras induced pathogenic myocardial hypertrophy.

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MATHERIALS AND METHODS

Reagents

HPLC-grade methanol (CH3OH) was from EM Science (Darmstadt, Germany). Acid cleavable detergent, 3-[3-(1,1bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), was purchased from Protein Discovery Inc. (Knoxville, TN). Ammonium bicarbonate (NH4HCO3), phenylmethylsulfonyl fluoride (PMSF), formic acid (HCOOH), and 2-iodoacetamide (IAA) were obtained from Sigma (St. Louis, MO). Tris[2carboxyethyl] phosphine (TCEP) Bond-Breaker and BCA assays were from Pierce (Rockford, IL). Fused-silica capillaries were acquired from Polymicro Technologies (Phoenix, AZ). All chemicals used were ACS grade or higher, and all solvents used were HPLC grade or higher. Sequencing grade trypsin was obtained from Promega (Madison, WI). All solutions were prepared using water purified with a Nanopure II system (Dubuque, IA). Transgenic Mice, Tissue Procurement, Homogenization, and Digestion

Mice with cardiac specific, tet-off controlled H-Ras-G12V expression used in this study were described previously.8 Briefly, two lines of transgenic animals were used. The first line, designated as M mice, carried the tTA gene controlled by the rat cardiac α-myosin heavy chain (MHC) promoter (Jackson Laboratory, Bar Harbor, ME). A second transgenic line, designated as R mice, has a mutated human H-Ras gene, HRas-G12V, under the control of a minimal promoter containing multimerized tet-operons (TetO) (NCI-Frederick, MD). Binary transgenic mice (M/R) were generated by crossing the two lines. The expression of H-Ras-G12V was regulated using dietary doxycycline. Disease induction was carried out by

Nanoflow Liquid Chromatography Mass Spectrometry

A total of four tissue slices (i.e., technical replicates) were obtained from two diseased hearts (i.e., two biological 1562



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replicates) and analyzed separately by LC-MS2 followed by equivalent analysis of four corresponding tissue slices obtained and prepared from two healthy/control hearts. Nanoflow reversed phase liquid chromatography (RPLC) MS analysis was performed using an Agilent 1100 nanoflow LC system coupled online with a hybrid linear ion trap (LIT)−Fourier transform ion cyclotron resonance (FTICR)-MS instrument (LTQ-FT, Thermo Electron, San Jose, CA). Reversed-phase columns (75 μm i.d. × 10 cm fused silica capillary with a flame pulled tip) were slurry-packed in-house with 5 μm of a 300 Å pore size C18 stationary phase (Phenomenex, Torrance, CA). After sample injection (1 μg of peptides), the column was washed for 20 min with 98% mobile phase A (0.1% formic acid in water) at a flow rate of 0.5 μL/min. Peptides were eluted from the column using a linear gradient of 2% mobile phase B (0.1% formic acid in acetonitrile (ACN) to 60% solvent B in 100 min at a flow rate of 0.25 μL/min, then to 98% B for an additional 10 min. Each sample (2 biological replicates) was analyzed separately in duplicate (4 technical replicates). The mass spectrometer was operated in a data-dependent mode to automatically switch between MS1 and MS2. The FT-ICR-MS1 survey scan (m/z range: 350 −1800) was followed by seven MS2 scans in which the most abundant peptide precursor ions detected in the preceding FT-ICR-MS1 survey scan were dynamically selected for collision induced dissociation (CID). The threshold of 200 ion counts was used for triggering an MS2 scan. The normalized CID energy was 35%; the electrospray voltage was set at 1.6 kV, and the voltage and temperature of the ion source capillary were set at 45 V and 160 °C, respectively.

run from Group B. The probability that the peptide ion comes from Group A is pA, and the probability that it comes from Group B is pB, where pA + pB = 1.0. If K peptide ions are observed from a given protein in both groups, the probability of a specific distribution of the K ions between the two groups is given by the binomial distribution (pA + pB)K. The null hypothesis assumes that there is no difference between the protein levels in Groups A and B, so the probability of finding a given peptide ion from A or B is the same (i.e., pA = pB = 0.5). If the number of observed peptide ions from Group A is less than the number from Group B, and this number is L, the probability that Group A will produce L ions or less from the situation where pA = pB is given by the following expression. P(L|K ) =

( ∑lL= 0 B(l , k)) 2K

In this expression, B(l,K) is the binomial coefficient for the pAl term in (pA + pB)K. Gene ontology term enrichment analysis was performed using DAVID bioinformatic software applied on a set of differentially expressed proteins.17 Cross-validation

Selected identified proteins were confirmed using IHC and/or WB as previously described.18 Both frozen tissue sections and aldehyde-fixed, paraffin-embedded tissue sections of myocardium were used for IHC. Antigen retrieval was performed for paraffin-embedded tissues by immersing tissues in target antigen retrieval buffer (TAR, Dako, Carpinteria, CA) in a steamer (Black and Decker, Hunt Valley, MD) for 15 min. Primary antibodies (see below) were followed by biotinylated goat antirabbit IgG secondary antibody (Dako, Carpinteria, CA). Immunoreactions were developed using a peroxidasebased streptavidin detection method (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Invitrogen Life Technologies, Carlsbad, CA) as chromogen substrate. Negative reaction control included substitution of antibody diluent for primary antibodies on tissue sections. Western blotting was used to analyze total tissue lysates from snap frozen ventricles. Tissues were disrupted in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% SDS, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, and 1 μg/mL leupeptin). Ten micrograms of clarified tissue protein lysates were separated on 4−20% Tris-Glycine gradient gels (Invitrogen Life Technologies), transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA), and blotted with primary antibodies followed by peroxidase-conjugated secondary antibodies. Primary antibodies used in IHC and WBs included the following: Ras, Wnt-3a, β-catenin, and vimentin (Abcam, Cambridge, MA); β-catenin, and GAPDH (Cell Signaling Technology, Danvers, MA). Biotin-labeled secondary antibodies used in IHC were from DakoCytomation (IHCCarpinteria, CA). Horseradish peroxidase-conjugated secondary antibodies for WB were obtained from Jackson ImmunoResearch (West Grove, PA).

Data Analysis and Bioinformatics

Acquired MS2 spectra from each technical replicate were searched independently against the mouse protein database (UniProt Mouse, release 09/2007), using SEQUEST (Thermo, San Jose, CA). The searches were carried out on a Beowulf 18node parallel virtual machine cluster-computer. Dynamic modification was added for the detection of oxidized methionine (+16 Da). For the MS1 spectra acquired by FTICR-MS, the monoisotopic precursor ion mass tolerance was set at 10 ppm Da, while for the data dependent MS2 spectra, acquired by LIT−MS, the fragment ion tolerance was set at 0.5 Da. Only fully tryptic peptides with up to two miscleavages possessing delta correlation ΔCn ≥ 0.1 and charge state dependent cross-correlation Xcorr ≥ 2.1 for [M + H]1+ ≥2.3 for [M +2H]2+ and ≥3.5 for [M + 3H]3+ were considered legitimately identified. Using these SEQUEST thresholds, the search against a decoy mouse database revealed a false-positive discovery rate at the peptide level of ≤5%.15 The final lists of protein identifications for diseased and control data sets were created by collating proteins identified in technical replicates as a nonredundant protein list where the given unique peptide identifies only a single protein. Relative differences in protein abundance between the diseased and control specimens were determined by calculating spectral count ratios.16 Relative changes in abundances for all proteins identified in both control and diseased specimens were computed using spectral counting that relies on strong correlations between relative protein abundance and the sum of all MS2 spectra/ peptides observed from a single protein.16 To determine the statistical significance of the changes in relative abundances for all proteins, p-values were calculated for all computed ratios on the basis of a pairwise comparison of a run from Group A and a

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RESULTS The aim of this study was a method-based investigation concerning the feasibility of using high-resolution and highaccuracy LC-MS to identify and quantify disease relevant proteins from tissue specimens obtained by employing a 1563



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standard histopathological protocol. Fresh-frozen myocardial tissue sections from transgenic mice with induced pathogenic myocardial hypertrophy (M/R mice, diseased myocardium) and healthy wild type mice (control/healthy myocardium) were used (i.e., two biological and four technical replicates each). Two biological replicates were chosen on the basis of the preliminary experiments using heart tissue obtained from nondiseased transgenic animals. Candidate proteins were selected using protein information generated from all technical replicates obtained from both diseased and healthy hearts, on the basis of results of the pathway meta-analysis. As previously established, M/R mice developed pathogenic myocardial hypertrophy relevant to human hypertrophic cardiomyopathy after the induction of H-Ras-12V expression.8 The histopathological findings of H&E stained ventricular tissue slices along with an increase of HW/BW ratios were utilized to validate the cardiomyopathy phenotype in animals used in this study (Supporting Information Figure 1). The experimental work flow is depicted in Figure 1. One thin fresh frozen heart tissue section mounted on a glass slide was scraped into 20% buffered methanol, followed by heat denaturation and trypsin digestion of the soluble proteins. In the next step, 60% buffered methanol was used to facilitate solubilization of hydrophobic/membrane protein species that are insoluble in 20% methanol.19 Equal amounts of trypsin digestates from diseased and control myocardium were analyzed in parallel using LC-MS. The optimization of upstream sample processing and assessment of reproducibility were carried out during preliminary experiments using tissue slices obtained from healthy hearts. We assessed that, by using four replicates, a reproducibility level of ∼75% was achieved, which is in agreement with previously published findings.20 The acquired MS2 spectra were searched independently against a mouse protein database using SEQUEST. Searching against a decoy mouse database revealed the false-positive discovery rate to be ≤5%.15 The final lists of protein identifications for diseased and control data sets were created by collating proteins identified in technical replicates. The relative change in protein abundance between control and diseased specimens was computed using spectral counting. This approach relies on strong correlations between relative protein abundances and the sum of all MS2 spectra/peptides observed from a single protein.16 Four hypertrophic heart specimens (technical replicates) yielded a total of 7290 peptides, of which 2763 were unique (Supporting Information Table 1A). These peptides led to the identification of 704 unique proteins (Supporting Information Table 1B), whereas Supporting Information Table 1C includes the protein IDs from each technical replicate. Likewise, using the same approach, a total of 6308 peptides was identified from control myocardial tissues. Of these, 2205 were unique (Supporting Information Table 2A), resulting in the identification of 752 unique proteins in control/healthy hearts (Supporting Information Table 2B). Supporting Information Table 2C contains the protein IDs from each technical replicate. A subtractive proteomic analysis21,22 was performed to compare data sets obtained from the hypertrophic and control hearts, respectively. The analysis allowed us to categorize the identified proteins into three groups: (i) 394 proteins that were exclusively identified in hypertrophic myocardium (Supporting Information Table 3A) but not in control myocardium; (ii) 442 proteins that were identified exclusively in control myocardium

Figure 1. Experimental design and flow chart depicting the processing of the standard fresh-frozen histopathological tissue slice employed in this analysis. The slice is mounted on a glass microscope slide routinely used in a clinical diagnosis. Tissue sections used in this study were unstained and generally 20 mm2 or less tissue area (depicted in the figure as stained with hematoxylin and eosin for purposes of illustration) and prepared as described in the Materials and Methods section. Equal amounts of trypsin digestates from diseased and control myocardium were analyzed in parallel using nanoflow reverse-phase LC coupled with a hybrid linear ion trap/Fourier transform−ion cyclotron resonance MS.

(Supporting Information Table 3B) but not in diseased myocardium, and (iii) 310 proteins identified in both diseased and control hearts (Figure 2A and Supporting Information Table 3C). An unsupervised cluster analysis was performed on the 310 proteins identified in both diseased and control hearts. This analysis revealed a subset of differently regulated proteins identified in both diseased and control hearts exhibiting statistically significant changes in their expression level. Of these, a subset of 28 proteins depicted in Figure 2B showed significant changes (p ≤ 0.05) in their abundances. In the diseased heart group, 25 proteins were up-regulated and three were down-regulated (Figure 2B). A DAVID analysis identified a significant functional annotation clustering of 14 proteins (Supporting Information Table 4) containing functional terms such as contractile fiber, myofibrill, and cytoskeleton.17 1564



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Table 1. Subset of Identified Proteins Directly Linked to Cardiovascular Disease by IPA ID

symbol

Entrez gene name

Proliferation/Survival Related Proteins P57110 ADAMTS8 ADAM metallopeptidase 8 P19091 Q02248 Q61411

AR CTNNB1 HRAS

P48678 P42227

LMNA STAT3

Q9CXZ1

NDUFS4

Q6IMG6 SETX P10649 GSTM5 Cytoskeletal Proteins P68033 ACTC1 P31001 DES Q9CV95 DSP

Figure 2. Number of proteins identified by MS following spectral count and subtractive analysis. (A) Venn diagram of number of proteins identified in diseased (M/R) and healthy (+/+) control hearts showing 310 proteins overlapped between the two groups. (B) Heat map and accession numbers of 28 protein species showing a significant difference in their relative expression levels based on spectral count statistical analysis of the 310 overlapping protein species identified in both diseased and healthy heart tissue.

Ingenuity pathway analysis (Ingenuity Systems, www. ingenuity.com) was performed to explore the relevance of this data set to cardiovascular disease and myocardial tissue in general. A total of 106 proteins for this analysis included proteins differentially expressed in diseased vs control hearts, as well as the proteins identified by ≥3 peptides in either the diseased or control hearts. The analysis revealed “cardiovascular disease, skeletal and muscular system development/function” as the most relevant networks, with 34 proteins directly involved in cardiovascular disorders (Table 1). Of these, 15 were part of the cytoskeleton and 10 were known to be middle to low abundance proteins involved in cell proliferation/survival. These proteins are highly relevant to the underlying hypertrophic myocardial pathophysiology and its associated phenotypes, including increased cell volumes and reactivated cell cycle regulatory proteins.

androgen receptor catenin, beta 1 v-Ha-ras, sarcoma viral oncogene lamin A/C signal transducer and activator of transcription 3 NADH dehydrogenase (ubiquinone) Fe−S protein 4 senataxin glutathione S-transferase mu 5 actin, alpha, cardiac muscle 1 desmin desmoplakin

P27546

MAP4

microtubule-associated protein 4 sorbin and SH3 domain containing 1 sorbin and SH3 domain containing 2 tubulin, alpha 1a tubulin, beta 2A vinculin

Q62417

SORBS1

Q80TS1

SORBS2

P68369 Q7TMM9 Q64727

TUBA1A TUBB2A VCL

A2ASS5 Q62261

TTN SPTBN1

Q8VIE5

SPTBN4

Q02566

MYH6

Q8VDD5

MYH9

Q02257

JUP

Q6P8P4

MYL2

P20152 Others Q8QZT1 P07356

VIM

titin spectrin, beta, nonerythrocytic 1 spectrin, beta, nonerythrocytic 4 myosin, heavy chain 6, cardiac muscle, alpha myosin, heavy chain 9, nonmuscle ã-catenin (junction plakoglobin) myosin, light chain 2, regulatory, cardiac, slow vimentin

ACAT1 ANXA2

acetyl-CoA acetyltransferase 1 annexin A2

Q45VK7

DYNC2H1

Q1MWP8

EHD4

dynein, cytoplasmic 2, heavy chain 1 EH-domain containing 4

P61014 P17742 P09671

PLN Ppia SOD2

Q04857

COL6A1

phospholamban peptidylprolyl isomerase A superoxide dismutase 2, mitochondrial collagen, type VI, alpha 1

subcellular location extracellular space nucleus nucleus plasma membrane nucleus nucleus cytoplasm nucleus cytoplasm cytoplasm cytoplasm plasma membrane cytoplasm plasma membrane nucleus cytoplasm cytoplasm plasma membrane cytoplasm plasma membrane cytoplasm cytoplasm cytoplasm plasma membrane cytoplasm cytoplasm cytoplasm plasma membrane cytoplasm plasma membrane cytoplasm cytoplasm cytoplasm extracellular space

A major goal of this method-based study was to determine if H-Ras could be identified by LC-MS in a fresh-frozen thin tissue section obtained from hypertrophic/diseased myocardium using a routine pathology procedure. Pathogenic myocardial hypertrophy was induced by cardiac-specific 1565



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Table 2. LC-MS Detected Peptides Identifying the H-Ras Precursor in Hypertrophic Heart reference

protein

peptide

z

XC

DelCn

Acc No

RASH_MOUSE RASH_MOUSE RASH_MOUSE

HRas precursor HRas precursor HRas precursor

K.TRQGVEDAFYTLVR.E R.QAQDLAR.S R.VKDSDDVPMVLVGNK.C

2 2 2

3.565 2.3223 3.4085

0.3713 0.1211 0.4956

Q61411 Q61411 Q61411

Vimentin was identified only in diseased hearts by 33 peptides, of which 16 were unique. Positive vimentin immunolabeling was observed in hypertrophic cardiomyocytes and in the increased fibroblasts present in the interstitium of diseased M/ R hearts (Figure 4A,B). As expected, positive vimentin staining in healthy control hearts was limited to blood vessel endothelium, myocytes, and occasional interstitial cells (Figure 4B). An increase in total vimentin level in diseased hearts was also observed in multiple specimens using immunoblotting (Figure 4C). The identification of induced H-Ras activation and the reactive cytoskeletal vimentin expression provides evidence that the MS analysis applied in this approach is adequate for interrogating histopathological thin fresh-frozen tissue sections. In addition to Ras and vimentin, β-catenin and Wnt-3a, two key players in Wnt signaling events, were unambiguously identified by LC-MS in diseased hearts, but not in healthy control hearts. This result was interpreted as an increased expression level of these two proteins during development of pathogenic myocardial hypertrophy, primarily due to their abundance levels below the MS detection limit in control/ healthy heart. Indeed, increased expression of β-catenin in multiple M/R hearts with advanced cardiomyopathy was validated by WB analysis (Figure 5A). β-Catenin IHC was carried out to further corroborate its elevated level with its activation status. Positive β-catenin immunolabeling in hypertrophic cardiomyocytes (Figure 5B), in contrast to cardiomyocytes in control hearts (Figure 5C), indicated that hypertrophic cardiomyocytes were the source of elevated β-catenin levels. In addition, there was notable nuclear accumulation of β-catenin in hypertrophic cardiomyocytes (Figure 5B), imparting evidence of β-catenin functioning as a transcriptional activator in hypertrophic cardiomyocytes. In contrast, β-catenin distributed at the intercalated disks near intercardiomyocyte junctions in healthy control hearts (Figure 5C). Similar localization of β-catenin at intercalated disks was markedly reduced in cardiomyopathic hearts (Figure 5B). The increased β-catenin level as well as its nuclear translocation indicated the activation of the β-catenin signaling pathway in pathogenic myocardial hypertrophy initiated by H-Ras activation. Next, LC-MS detection of Wnt-3a in hearts with pathogenic myocardial hypertrophy was validated using IHC. Increased labeling intensity was observed in hypertrophic cardiomyocytes in M/R hearts compared to healthy control hearts (Figure 6A,B). Similarly, increased intensity of phospho-GSK-3β immunolabeling was detected in hypertrophic cardiomyocytes while the background level was seen in healthy control hearts (Figure 6C,D). Increased Wnt-3a expression may have resulted in the activation of the canonical Wnt signaling pathway and therefore set up the environment for the subsequent activation of β-catenin. The preanalytic sample preparation as well as data interrogation approach provided noteworthy evidence of the capability of this approach to detect disease-relevant cell signaling proteins, which are normally present in relatively low

expression of a constitutively active H-Ras mutant, H-RasG12V. Three unique H-Ras peptides were detected by LC-MS in hypertrophic myocardium but not in control mouse myocardium (Table 2). Multiple tissue sections obtained from the same specimens used in MS analysis, as well as from hearts of additional M/R and control mice, were used to cross-validate MS findings. IHC evidence of increased Ras levels in hypertrophic cardiomyocytes was observed (Figure 3A). Western blotting

Figure 3. Validation of Ras expression. Ras IHC on tissue sections from the original (M/R) diseased/hypertrophic (A) and healthy (WT) control (B) myocardial specimens used in the MS analyses. Representative IHC results on tissue sections are shown. Positive immunolabeling is depicted as brown chromogen, with hematoxylin counterstain. Bar = 50 μm (C) WB analysis of Ras expression level in additional wild type (WT) control and diseased (M/R) hearts. Assigned lesion scores for each specimen are indicated (0 = no disease, 3 = severe disease).

was used to further corroborate the MS and IHC results in additional specimens with the same disease induction duration and similar range of myocardial lesion scores. The level of Ras protein correlated with the predetermined lesion scores, indicating a causal role of H-Ras-G12V induction in the disease model (Figure 3C). The detection of H-Ras by LC-MS was considered indispensable for confident discrimination between the diseased and control (nondiseased) molecular phenotypes. Vimentin was also identified, as one of the hallmark proteins elevated in pathogenic myocardial hypertrophy, due to a responsive increase in cardiomyocyte intermediate filaments, coupled with fibroplasia in pathogenic myocardial hypertrophy. 1566



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Figure 5. Validation of β-catenin expression. (A) WB analysis of multiple healthy wild type (WT) control and diseased (M/R) hearts showed an increase of total β-catenin expression level in diseased hearts. The assigned lesion scores for each specimen are indicated (3 is most severely affected). β-catenin IHC in hypertrophic M/R hearts (B) further demonstrated increased cytoplasmic labeling (red arrow) and nuclear accumulation (black arrows). This was associated with sporadic intercalated disk localization of β-catenin (black arrowhead) in hypertrophic cardiomyocytes. In control hearts (C), however, intercalated discs were the primary site of β-catenin immunolabeling (black arrow).). Representative IHC results on tissue sections are shown here, with positive immunolabeling depicted as brown chromogen, hematoxylin counterstain. Bar = 50 μm.

Figure 4. Validation of vimentin expression. IHC for vimentin was carried out in hypertrophic M/R and control (WT) hearts. In the hypertrophic heart, positive immunolabeling was observed in cardiomyocytes (black arrows) as well as interstitial fibroblasts (red arrows) and endothelial cells (green arrows) (A). In the healthy control heart (B), endothelial cells are the major cell type labeled (red arrow). Representative IHC results on tissue sections are shown here, with positive immunolabeling depicted as brown chromogen, hematoxylin counterstain. Bar = 50 μm. (C) WB analysis of vimentin expression level in additional wild type (WT) control and diseased (M/R) hearts. Assigned lesion scores for each specimen are indicated (0 = no disease, 3 = severe disease).

typically in limited supply, often due to their procurement by minimally invasive techniques and requirements for their use in diagnostics. In this study, a two-stage methanol assisted protein solubilization/digestion method was applied for the MS analysis of frozen tissue sections mounted on glass microscope slides, similar to those used for histopathological diagnosis.26 Sufficient amounts of protein for LC-MS analysis were obtained from as little as a single 8 μm thick tissue section. Subsequent pathway analysis revealed a proteome highly relevant to the disease model. Thus, this approach provides a means to analyze whole lysates of heart tissue sections, in a manner directly analogous to the current standard pathological tissue slide analysis, used for routine clinical diagnosis. The discovery and identification of disease-relevant proteins in vivo as well as the further validation of candidate biomarkers are fundamental to the value of MS-based proteomics. Using

abundance, compared to the overexpressed H-Ras and cytoskeletal proteins.

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DISCUSSION Specimen procurement, preservation, and processing are fundamental steps in the proteomics workflow.23−25 Discoveries of candidate biomarkers rely upon identifying disease relevant proteins from clinical tissue specimens that are 1567



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overexpression of the myosin heavy chain cardiac muscle beta isoform in a diseased ventricle (Supporting Information Table 3C) are in agreement with previous findings.8,28 In this study, the MS-based proteomic profiling of thin fresh frozen tissue sections revealed Wnt/β-catenin activation secondary to Ras signaling. Concomitant detection of Wnt3a and β-catenin suggested the reactivation of this fetal program and strongly implicated the significance of this pathway during cardiomyopathy disease progression. Furthermore, the increased β-catenin protein level was accompanied by its translocation into cardiomyocyte nuclei. This was demonstrated by IHC and provides additional indication of β-catenin transcriptional activation. While Wnt activation plays a significant role in β-catenin transcriptional activation in pathogenic myocardial hypertrophy,30 other factors may also be involved in influencing βcatenin signaling events. Concurrent increases in inactive GSK3β (phosphorylated GSK-3β) in hypertrophic cardiomyocytes suggested that increased β-catenin levels could be contributed, at least partially, to the inhibition of its degradation.29 In addition to activation of the canonical Wnt pathyway, the phosphorylation of GSK-3β can also be achieved by several kinases, such as ERK and AKT.31,32 We have previously reported that ERK1/2 is the major MAPK activated upon HRas-G12V induction in Ras-induced pathogenic myocardial hypertrophy.8 It is conceivable that both Wnt and ERK contributed to GSK-3β inactivation at this disease stage. Since AKT activation was not detected in this model,8 it seems unlikely that PI3K/Akt was associated with the observed increased phosphorylation of GSK-3β. Not only is β-catenin part of a catenin/T-cell factor (TCF) transcription complex in the nucleus, it is also a part of cadherin/catenin complexes at cell−cell junctions, making it a dual function protein.33,34 Increased β-catenin levels have been observed at cardiomyocyte intercalated discs in hypertrophic cardiomyopathy.35 Intercalated discs are crucial in transmitting electric and mechanical signals between cardiomyocytes. Disruption of intercalated disk structures has been observed in cardiomyopathies and other cardiac dysfunctions.36 Our findings demonstrated altered distribution of β-catenin in cardiomyocyte junctional complexes, which was coupled with overall increased myocardial β-catenin levels. These findings are in concert with others who have similarly documented the development of pathological myocardial hypertrophy under these circumstances.37 Several other proteins associated with cardiomyocyte intercalated discs, such as γ-catenin, vinculin, and desmoplakin, were found to be significantly upregulated in hypertrophic heart (Figure 2). In β-catenin deficient mice, γcatenin has been shown to substitute for β-catenin in Ncadherin binding,38 perhaps in an attempt to compensate functionally. Whether this dissociation of β-catenin from its normal location within intercalated discs, along with elevated γcatenin levels, are the cause of or result from cytoskeletal structural alterations in hypertrophic cardiomyocytes remains undetermined. Advanced stages of pathogenic myocardial hypertrophy were characterized not only by the presence of hypertrophic cardiomyoctyes but also by fibroplasia, to repair cell loss. Increased vimentin levels have been associated with the transition to heart failure,39 and its increase has been documented in interstitial cells (in failing human hearts)40 as well as in cadiomyocytes (in dilated cardiomyopathy).41 Significant overexpression of vimentin intermediate filament

Figure 6. IHC of Wnt-3a and pGSK-3β in serial tissue sections. Wnt3a IHC revealed an increased Wnt-3a level in hypertrophic M/R cardiomyocytes (A), in contrast to the even background labeling observed in control hearts (B). Likewise, increased pGSK-3β immunolabeling was observed in hypertrophic cardiomyocytes (C), shown in serial tissue sections from the specimen illustrated in part A, whereas background labeling was seen in control hearts (D). Representative IHC results on tissue sections are shown here, with positive immunolabeling depicted as brown chromogen, hematoxylin counterstain. Bar = 50 μm.

the animal model of pathogenic myocardial hypertrophy, the validity of the proteins identified through this approach was established in two complementary ways. First, genetic control of H-Ras activation in transgenic mice led to pathogenic myocardial hypertrophy with human disease-like characteristics, which permitted study of individual known reference proteins, such as H-Ras, altered during cardiac disease. Second, the experimental model provided a platform to test the feasibility of using MS to directly analyze the cardiac tissue proteome while delineating the role of H-Ras activation during the initiation and progression of hypertrophy. The identification of unique peptide sequences from H-Ras and other middle to low range expressed signaling molecules, such as Wnt-3a and β-catenin, demonstrated the utility of this approach. Specifically our findings associated two signaling events, Ras activation and Wnt/β-catenin signaling, with pathogenic myocardial hypertrophy progression. The fact that protein lysates were derived from hearts with advanced cardiac lesions, representing M/R mice with functional evidence of heart failure observed both clinically and pathologically, provided support for the relevance of the findings to human cardiomyopathy. The canonical Wnt/β-catenin signaling pathway initially becomes activated and then inactivated during cardiac development.27 Thus, it is considered to be a part of a “fetal gene program” that is turned off when cardiomyocyte development is completed shortly after birth.28 Pathogenic myocardial hypertrophy involves reactivation of the fetal genes, such as the atrial natriuretic factor (ANF) and β-MHC.29 This reactivation was previously demonstrated in the H-Ras model during induction of cardiomyopathy.8 The identifications of the myosin light chain 1, atrial/fetal isoform and myosin regulatory light chain 2, atrial isoform in the diseased ventricle only (Supporting Information Table 3A) along with observed 1568



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proteins in hypertrophic M/R myocardium was observed by relative MS quantitation. This finding was also validated by IHC and immunoblots, providing additional evidence for the relevance of the methods developed in this study. Previously we described approaches for proteomic profiling of amount-limited tissue specimens collected by laser capture microdissection (LCM) as well as for in-depth profiling of substantially larger tissue specimens using advanced multidimensional shotgun proteomics.22,26 Both of these strategies rely on sophisticated analytical technologies and expensive equipment (e.g., LCM apparatus, advanced LC systems) commonly not accessible to all research laboratories. However, the method illustrated in this study does not require complicated tissue processing and fits seamlessly into routine pathology work-flows. A simple LC-MS method capable of comparative differential proteomic profiling of thin fresh frozen tissue sections has been presented in this study. The use of subtractive proteomics and label-free quantitation permitted advanced proteome-wide molecular characterization of the H-Ras induced myocardial hypertrophy by identifying principal proteins driving the pathological process. This is the first MS-based proteomic study demonstrating the activation of the Wnt/β-catenin pathway during the pathogenic myocardial hypertrophy, secondary to constitutive H-Ras activation. The LC-MS based elucidation of key proteins, cross-validated by WB and IHC, may be subsequently investigated using complex immunoassays (i.e., ELISA) in a high-throughput manner in multiple blood specimens if the proteins of interest, previously characterized in tissue, are expected to leak into the peripheral circulation or other body fluids. We anticipate that the proteomic approach described herein may be applied to many different tissue types for in vivo molecular profiling and improve the understanding of pathology under study.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Tables of peptide identifications and images of H&E stained tissue sections. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 301-846-7211. Fax: 301-846-6037. E-mail: blonderj@ mail.nih.gov.

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ACKNOWLEDGMENTS This research was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute (NCI), Bethesda, MD. Additional support was provided with federal funds from the National Cancer Institute, National Institutes of Health, under Contract N01-CO-12400. H.R.S. was a molecular pathology fellow in the NIH Comparative Biomedical Scientist Training Program supported by the National Cancer Institute in partnership with the University of Maryland. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government. 1569



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