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Hypoplastic left heart syndrome myocytes are differentiated but possess a unique phenotype
Cardiovascular Pathology 12 (2003) 23 – 31
Hypoplastic left heart syndrome myocytes are differentiated but possess a unique phenotype Teresa J. Bohlmeyer, Steve Helmke, Shuping Ge, Jennifer Lynch, Gary Brodsky, James H. Sederberg II, Alastair D. Robertson, Wayne Minobe, Michael R. Bristow, M. Benjamin Perryman* Department of Medicine, Division of Cardiology, University of Colorado Health Sciences Center, Denver, CO, USA Department of Pediatrics (SG), University of Colorado Health Sciences Center, Denver, CO, USA Received 5 February 2002; received in revised form 15 July 2002; accepted 22 July 2002
Abstract Introduction: Hypoplastic left heart syndrome (HLHS) is the term used to describe a group of congenital malformations characterized by marked underdevelopment of the left side of the heart. HLHS accounts for nearly 25% of cardiac deaths in the first year of life. Although much has been reported regarding diagnosis, gross morphology and surgical treatment, no information on gene expression in HLHS myocytes is available. Methods: We examined heart tissue from patients with HLHS using routine histology, immunohistochemistry, quantitative polymerase chain reaction (PCR), two-dimensional (2-D) gel electrophoresis and protein identification by mass spectrometry. Results: Histologic examination of right and left ventricles from HLHS patients revealed characteristic features of myocyte differentiation, including striations and intercalated disc formation. Immunohistochemical staining using antibody to N-cadherin demonstrated clear development of intercalated discs between myocytes. However, many of the myocytes contained scant cytoplasm and were grouped in small, disorganized bundles separated by abundant connective tissue and dilated, thin-walled vessels. Quantitative PCR analysis demonstrated that both left and right ventricular tissue from HLHS hearts expressed the fetal or ‘‘heart failure’’ gene expression pattern. Two-dimensional gel electrophoresis and protein identification by mass spectrometry also confirmed that myocytes from HLHS ventricles were differentiated but expressed the fetal isoform of some cardiac specific proteins. However, HLHS myocytes in all of the heart samples (n = 21) were inappropriately expressing platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), a member of the cell adhesion molecule (CAM) family that has a primary role in the regulation of tissue morphogenesis. These findings indicate that myocytes from HLHS syndrome patients, while differentiated, have a unique gene expression pattern. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Hypoplastic left heart syndrome; Myocytes; Gene expression; Immunohistochemistry; Mass spectrometry
1. Introduction The term hypoplastic left heart syndrome (HLHS) is used to describe a group of malformations characterized by marked underdevelopment of the left side of the heart. The anatomic abnormalities include underdevelopment of the left atrium and ventricle, stenosis or atresia of the aortic or mitral orifices, and marked hypoplasia of the ascending aorta [1,2]. HLHS accounts for approximately 7– 8% of heart disease producing symptoms in the first year of life * Correspondence author. Department of Medicine, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B139, Denver, CO 80246, USA. Tel.: +1-303-315-3259. E-mail address: [email protected] (M.B. Perryman).
and is the most common cause of death from heart disease in the first week after birth [3]. It accounts for nearly 25% of cardiac deaths in the first year of life (New England Regional Infant Cardiac Registry). The prognosis for most patients with HLHS is bleak and treatment is surgery to repair the defects or, more rarely, cardiac transplantation. HLHS is unlike most other complex congenital heart diseases in that abnormalities in other organ systems are rare. Thus, children with HLHS are, except for heart abnormalities, normal [4]. The genetic basis of HLHS is not clear, although in some cases the syndrome has been reported to be associated with chromosomal abnormalities [5,6]. Microscopic examination of tissue sections from both right and left ventricles of patients with HLHS shows disorganized, variably sized bundles of myocytes coursing
1054-8807/02/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 4 - 8 8 0 7 ( 0 2 ) 0 0 1 2 7 - 8
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through abundant loose connective tissue and scattered, dilated, thin-walled blood vessels. Myocytes are focally small with scant cytoplasm. It is not known if HLHS myocytes express proteins typical of differentiated myocytes or remain significantly undifferentiated. Platelet-endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily [7]. The 130-kDa fulllength protein possesses six extracellular immunoglobulin domains, a transmembrane portion and a cytoplasmic domain [8]. Functionally, PECAM-1 is an adhesion molecule with both homophilic and heterophilic binding. The homotypic binding is important in leukocyte transendothelial migration [9]. The heterotypic ligands have been reported to include integrin, avb3 [10] and glycosaminoglycans [11]. PECAM-1 is present on the surface of platelets, some white blood cells and endothelial cells [7,12], and is strongly expressed by all endothelial cells and to a lesser extent on several types of leukocytes [13]. Immunohistochemical staining for PECAM-1 is routinely used to demonstrate angiogenesis in several types of cancer [14 –19]. Protein expression studies have demonstrated that PECAM-1 is leukocyte and endothelial cell-specific and that PECAM-1 is not expressed in mouse cardiac myocytes at any time during development or in any cardiac diseases [20 – 22]. However, Hwang et al. reported expression of PECAM-1 in 8– 12-week fetal human hearts but not in older fetal or adult hearts. Since this analysis was cDNA-based, it cannot be determined if PECAM-1 mRNA expression in fetal hearts of this age is localized to myocytes or other cell types within the myocardium [23]. In addition, PECAM-1 null mice have no discernible cardiovascular malformations and therefore PECAM-1 expression is not necessary for normal cardiovascular development in the mouse [24]. With improvements in early diagnosis of HLHS and the increasing potential for surgical intervention in the fetus, it is essential to determine the pathophysiological basis for the ventricular malformations and to determine the potential for HLHS myocytes to respond to normalized ventricular flow. If the ventricular myocytes are unable to respond appropriately following surgical correction, early intervention may be futile. We have used immunohistochemistry, quantitative reverse transcription polymerase chain reaction (QRTPCR) and two-dimensional (2-D) gel electrophoresis coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to determine that HLHS left ventricular myocytes have a differentiated but unique phenotype. The myocytes express mRNAs and proteins characteristic of differentiated, failing cardiac myocytes and form striations and intercalated discs. However, HLHS myocytes in all patient samples (n = 21) inappropriately express PECAM-1, a member of the CAM family that has a primary role in the regulation of tissue morphogenesis. Cardiac myocytes have never been shown to express PECAM-1 at any stage of development or in any disease state. These findings indicate that myocytes from HLHS
syndrome patients, while differentiated, have a unique gene expression pattern.
2. Methods 2.1. Tissue procurement Explanted human heart tissue was obtained during heart transplantation from pediatric HLHS patients (n = 21, ages 6 days to 10 months), nonsuitable pediatric donors (n = 2, ages 18 months and 8 years), pediatric transposition of the great vessels (n = 1, age 20 years), mitral stenosis (n = 1, age 4 years), idiopathic dilated cardiomyopathy (IDC; n = 2, ages 1 and 10 months), transplant rejection (n = 1, age 9 years), myocarditis (n = 1, age 2 months), hypoplastic right heart (n = 2, ages 2 and 5 months), adult patients transplanted for heart failure (n = 4, ages 18 – 64 years), including an 18-year-old male with IDC, and adult nonfailing, nonsuitable donors (n = 2, ages 28 and 45 years). Tissue was taken from both the right and left ventricular free wall, when available. All patients had been taking a variety of drugs, primarily diuretics, immunosupressants and positive inotropes. 2.2. Histology Heart tissue was formalin-fixed, routinely processed, paraffin-embedded, cut to 5-mm-thick sections onto Fisherbrand superfrost/Plus slides, and stained with hematoxylin and eosin and trichrome. 2.3. Immunohistochemistry Immunohistochemical detection was performed using the labeled avidin –biotin (LAB) system (DAKO LSAB + kit, peroxidase), monoclonal mouse antihuman endothelial cell, CD31 (PECAM-1) antibody (DAKO) and polyclonal rabbit antimouse, rat, human N-cadherin (Santa Cruz Biotechnology). Briefly, 5-mm-thick sections of formalin-fixed, paraffin-embedded heart tissue were deparaffinized in xylene, rehydrated and microwaved in 10-mM citrate buffer for antigen retrieval. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide. Appropriately diluted primary antibody was applied, followed by the biotinylated universal (swine antigoat, mouse, rabbit) secondary antibody and finally peroxidase-labeled streptavidin, which binds to biotin. Appropriate blocking steps using dilute bovine serum albumin, nonimmune swine serum and biotin – avidin were performed. The color reaction was developed using DAB (3,30-diaminobenzidine), resulting in a reddish-brown precipitate at the antigen site. Sections were counterstained with hematoxylin. Negative controls using appropriately diluted nonimmune serum instead of primary antibody were performed for each experiment. Endothelial cells lining blood vessels in each tissue section served as positive controls.
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2.4. Quantitative PCR Using a quantitative PCR technology developed within our group [25,26], we examined the expression of several fetal genes, b1- and b2-adrenergic receptors (AR), a- and b-myosin heavy chain (MHC), atrial natriuetic peptide (ANP) and sarcoplasmic reticulum calcium ATPase (SRCA), and compared their expression to that of adult nonfailing myocardium. The detailed methodology and values for the adult heart measurements have been previously reported [26]. 2.5. 2-D gel electrophoresis Approximately 50 mg of ventricular tissue was homogenized on ice using a ground glass homogenizer in 500 ml of sample buffer I from Genomic Solutions (GSI), 50 mM Tris, pH 8.0, 0.2 M DTT, 0.3% SDS supplemented with a protease inhibitor cocktail of 1 mM AEBSF, 0.1 mM leupeptin and 10 mg/ml aprotinin, all from Sigma. A 50-ml aliquot of sample buffer II (GSI; 0.5 M Tris, pH 8.0, 50 mM MgCl2, 2000 units/ml DNAse I, 750 units/ml RNAse A) was added and the mixture incubated on ice for 10 min to digest nucleic acids. The proteins were quantitatively precipitated by an adaptation of the method of Wessel and Flugge [27]. To the approximately 600 ml of homogenate was added methanol:chloroform (4:1, 750 ml) and the mixture was vortexed vigorously at room temperature. (All subsequent steps were performed at room temperature.) The mixture was centrifuged at 16,000 g for 5 min to separate phases, and the upper aqueous phase was carefully removed so as not to disturb the protein disc at the interphase. Methanol (450 ml) was added, and the sample thoroughly mixed to give a single phase. The protein was pelleted by centrifugation (16,000 g, 5 min) and the supernatant removed. The protein pellet was then dried in a Speedvac for 30 min. IEF sample buffer (500 ml) was added to each pellet; the samples were incubated overnight at room temperature. IEF sample buffer was prepared from IPG strip rehydration buffer (GSI), 8 M urea, 2% CHAPS, 2% ampholytes by supplementing it with 2 M thiourea and 50 mM DTT. After incubation, vigorous mixing and homogenization with a pellet pestle (Fisher) dispersed any undissolved material. Centrifugation (16,000 g, 10 min) pelleted unsolubilized material, mainly connective tissue. The supernatant was collected and a 20-ml aliquot was diluted 1– 50 with water and assayed for protein by the method of Bradford [28] (BioRad Protein Assay). IPG strips (18 cm, nonlinear pH 3– 10, GSI) were rehydrated with 2.5-mg protein in 400 ml of IEF sample buffer. After overnight rehydration in a humidified chamber, the IPG strips were subjected to 100,000 V h of focusing over 24 h. The strips were equilibrated for 10 min in equilibration buffer I (GSI) and for 10 min in equilibration buffer II (GSI) to reduce and alkylate the proteins with iodoacetamide. The strips were then placed on precast 10% polyacrylamide tricine double gels (GSI) and electrophoresed at 14 W/gel
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for 6 h. The gels were stained with colloidal Coomassie (Invitrogen) and destained with water. Images of the gels were captured with a Umax scanner. 2.6. In-gel tryptic protein digest Protein spots were excised from the gels with a truncated 1-ml pipet tip. Gel pieces were washed 2 for 10 min with 100 ml 50% acetonitrile (CH3CN)/25 mM ammonium bicarbonate (NH4HCO3) and then once with 100 ml 100% CH3CN. The gel pieces were dried in a Speedvac for 15 min. Promega sequencing grade trypsin (20 ml of 20 mg/ml in 10 mM NH4HCO3) was added and the gel pieces were allowed to rehydrate on ice for 20 min, incubated overnight at 37. The next day the peptides were extracted into 50 ml of 50% CH3CN/5% trifluoroacetic acid (TFA) with vigorous mixing (1 h). The extracts were transferred to fresh tubes and taken to dryness (Speedvac). The peptides were then redissolved in 0.5% TFA (20 ml). ZipTips (C18, 0.6 ml bed volume, Millipore) were wetted with 50% CH3CN and equilibrated in 0.1% TFA. Peptides were bound by pipetting 10 through the bed. The tips were then washed 2 with 0.1% TFA. Peptides were eluted with 50% CH3CN/0.1% TFA (5 ml) by pipetting 5 through the bed. 2.7. Mass spectrometry and protein identification The peptide eluate (0.5 ml) and matrix solution (0.5 ml) were thoroughly mixed on a clean stainless steel MALDITOF MS target and allowed to dry. The matrix solution was 10 mg/ml a-cyano-4-hydroxycinnamic acid dissolved in 50% CH3CN/0.1% TFA. A standard mix of bradykinin, angiotensin, glu-fibrino-peptide and ACTH 18 – 30 in matrix was also applied to an adjacent region of the target. MALDI-TOF spectra were acquired on a Voyager-De Pro (PerSeptive Biosystems) instrument operating in reflector mode. Following initial data collection using external calibration on the standard mix, the spectra of unknowns were recalibrated using autolytic tryptic fragments as internal mass markers. Mono-isotopic peptide masses with a signal to noise of greater than 5:1 were entered into the Protein Prospector MS-Fit program (www.prospector.ucsf.edu). This program finds the best fit of observed tryptic peptides to predicted tryptic peptides from known proteins [29]. These masses were searched at a tolerance of 0.1 Da against the SwissProt database. Cysteine carbamidomethylation was permitted as a modification to account for alkylation by iodoacetamide. Oxidation of methionine was also permitted because this has been observed after colloidal Coomassie staining. In the case of highly conserved proteins such as actin the search was limited to Homo sapiens. Our criteria for the positive identification of a protein included that it be the top MSfit candidate, that its MOWSE score be severalfold higher than the next best candidate, that the majority of peptides matched this protein and that at least six peptides matched this protein. The predicted molecular weight (MW)
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and pI were also considered in making the identification (Table 2). 2.8. Western blotting Total protein (30 mg) was separated on a 7.5% polyacrylamide gel, transferred to nitrocellulose membrane, probed with a 1:1000 dilution of anti-PECAM-1 antibody and detected using enhanced chemiluminescence (ECL) (Renaissance NEN). The image of the Western blot was captured and densitometry analysis performed using a SUN workstation.
3. Results 3.1. Histopathology Hematoxylin and eosin and trichrome-stained sections of HLHS ventricles revealed similar histologic features in both right and left ventricles. All sections contained large areas of randomly oriented, disorganized bundles of myocytes, with variability of myocyte size found between bundles. Some bundles were composed of normally proportioned and sized myocytes with centrally located, ovoid nuclei; others contained crowded, thin myocytes with scant cytoplasm and an increased nuclear to cytoplasmic ratio. Abundant loose connective tissue was found separating myocyte bundles and, focally, surrounding individual myocytes. Large, thinwalled ‘‘sinusoids’’ were observed scattered throughout the tissue, surrounded by more dense fibrous tissue. Even in areas containing normal-sized myocytes with only thin fibrous bands separating myocyte bundles, the individual myocytes appeared randomly oriented and disorganized. In some patients, the histologically abnormal areas were greater in the right ventricle than in the left. Many of these
Fig. 2. N-Cadherin immunostaining. Immunohistochemical demonstration of intercalated discs by detection of N-cadherin. Striations are also evident (LV from a different 3-month-old HLHS patient than shown in Fig. 1, N-cadherin immunostain, original magnification 150).
features can be seen in the trichrome-stained HLHS left ventricular section shown in Fig. 1. 3.2. N-Cadherin expression Immunohistochemical detection of N-cadherin revealed clear development of intercalated discs between myocytes even in areas of marked disorganization and abnormal myocyte morphology. Striations are also clearly seen (Fig. 2). 3.3. mRNA and protein expression One of the characteristics of failing myocardium is the reactivation of expression of a number of fetal genes coupled with down-regulation of a number of genes encoding contractile function regulating proteins in the adult ventricle. Using quantitative PCR technology, we examined left ventricular tissues from five HLHS patients and two normal pediatric donor hearts for expression of some of these genes. The mean values for HPLS measurements reported as mRNA molecules 105/mg of total mRNA is shown in Table 1. The P-values and ratios for all measurements are also shown. HLHS left ventricle mRNA expression of b1-AR, a-MHC, b-MHC and SERCA mRNA concentrations are reduced when compared to the normal left ventricles. The mRNA concentrations for ANF and Table 1 mRNA abundance in HLHS ventricles
Fig. 1. Hypoplastic left heart syndrome. Disorganized bundles of myocytes, large areas of fibrosis and abnormal vascular formations characterize both left and right ventricles in patients with HLHS (LV from 3-month-old HLHS patient, trichrome stain, original magnification 25).
mRNA
HLHS LV mean
Donor LV mean
P-value
Ratio
b1AR b2AR aMHC bMHC ANP SERCA
0.28 2.40 1.88 36.59 27.97 75.23
0.45 2.36 17.70 59.41 27.89 206.47
.33 1.00 .08 .18 1.00 .08
0.62 1.02 0.11 0.62 1.00 0.36
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Fig. 3. Colloidal Coomassie stained 2-D gels of adult (IDC) and pediatric (HLHS) human heart. Left ventricular tissue was prepared as described in Section 2 and the proteins resolved by 2-D gel electrophoresis. Proteins are identified by peptide mass mapping as described. Note the similarity in pattern between the pediatric and adult heart proteins. The atrial light chain 1 is an embryonic form expressed in the pediatric ventricle that disappears with maturity, but is reexpressed at a low level in failing adult human heart.
b2-AR are essentially unchanged as compared to normal controls. These data indicate that the HLHS left ventricles express at least a portion of the heart failure or fetal gene expression pattern. Even more importantly, these data clearly indicate that the HLHS ventricles are comprised of differentiated myocytes expressing the cardiac myocyte specific genes, SERCA 2a, a-MHC and ANF. Since mRNA concentrations do not necessarily correlate with the concentration of the corresponding protein we used 2-D gel electrophoresis coupled with protein identification by MALDI-TOF MS to compare HLHS ventricular protein expression to normal and failing ventricular protein expression. The protein expression profile of HLHS pediatric LV was compared to that of failing (IDC) adult LV using highresolution large format 2-D gel electrophoresis (Fig. 3). The protein spot pattern of the HLHS pediatric LV was very similar to that of the adult LV. While there were some differences in the minor spots, all the major spots were in the same position and had the same intensity on both gels,
with one exception, the expression of ALC-1 in the HLHS but not in the normal left ventricle. This indicates that the protein expression of the HLHS pediatric LV is very similar to the adult LV and that the HLHS ventricle is composed of differentiated myocytes. In order to identify the major protein spots and to confirm that the same proteins were expressed in both HLHS pediatric LV and adult LV, the spots were excised from both gels, digested with trypsin and analyzed by MALDI-TOF MS. Spectra were acquired in the highresolution reflector mode and the autolytic tryptic fragments were used as internal mass markers to increase mass accuracy. For each peptide, the mono-isotopic mass was identified and recorded for database searches. The SwissProt database was searched at a tolerance of 0.1 Da using Protein Prospector MS-Fit. Protein identification required a high MOWSE score relative to other candidates, matching a majority of the peptides, matching at least six peptides and an experimentally determined MW and pI consistent
Table 2 Proteins identified by peptide mass matching, MW and pI Protein
Theoretical MW
Experimentally determined MW
Theoretical pI
Experimentally determined pI
Peptides matched/submitted
% AA coverage
Tropomyosin ALC-1 Actin Troponin T M-CK VLC-2 VLC-1 Fatty acid binding-protein a-Crystallin B-chain Myoglobin
32,709 21,565 42,019 34,591 43,102 18,790 21,932 14,858 20,159 17,184
33,000 25,000 42,000 40,000 43,000 19,000 22,000 15,000 21,000 17,000
4.69 4.97 5.23 5.13 6.77 4.92 5.03 6.29 6.76 7.14
4.7 5.4 5.6 5.6 6.4 5.3 5.5 6.2 6.6 7.1
11/12 9/9 10/15 9/9 10/12 11/12 9/11 5/10 7/13 6/7
30 46 30 32 29 54 44 35 32 44
Following in-gel digestion with trypsin, proteins are identified by peptide mass matching. Identifications are confirmed by comparing the theoretical MWs and pI’s of these proteins with the MWs and pI’s experimentally determined from their position on the 2-D gels. The number of peptides matched to the predicted protein over the number of peptides submitted to MSfit is shown. The percent coverage is the number of amino acids in the matched peptides as a percentage of the total amino acids in the predicted protein.
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with the predicted values (Table 2). The experimentally determined MWs matched very closely to the predicted MWs except for atrial light chain-1 (ALC-1) and troponin T, which had slightly lower mobilities than expected. The experimentally determined pI’s were all within 0.5 pH units of the predicted values. (Actual pI’s are modified by charge distributions and interactions, and will often vary from theoretical pI’s, which are estimated from amino acid composition.) ALC-1 is calculated to have almost the same MW and pI as VLC-1, but appears somewhat larger and more acidic than VLC-1. This has been observed in other 2-D gel studies of human heart [30,31]. ALC-1 is significantly less abundant in the adult heart and is the embryonic isoform of LC-1 in the ventricle. During development, there is a switch in the ventricle from ALC-1 to the adult isoform VLC-1. In the pediatric heart, this switch is about half complete. The adult ventricle expresses almost exclusively VLC-1; but, in heart failure, it has been reported that there is re-expression of the embryonic isoform, ALC-1 [30,31]. The adult heart shown in Fig. 3 is a failing heart from a patient with IDC and this expression of ALC-1 probably reflects the fetal gene program. The major spots identified in Fig. 3 represent some of the most abundant proteins of the heart. As expected these include the contractile apparatus proteins actin, tropomyosin, myosin light chains and troponin T. Major metabolic proteins include myoglobin, fatty acid binding protein and M-CK. The most abundant member of the small heat shock family protein in heart is a-crystallin B-chain is also present. Analysis of the protein expression pattern of demonstrates that the myocytes of the HLHS ventricle are in a differentiation state similar to adult ventricular myocytes. 3.4. PECAM-1 expression Examination of sections of right and left ventricle used for immunohistochemical detection of PECAM-1 from 21 children transplanted for HLHS showed a distinct brown staining of endothelium lining both the large and small blood vessels. In addition, patchy and, in some patients, extensive areas of all sections examined contained distinct brown staining within myocytes, particularly in areas in which the myocytes appeared smaller, with less cytoplasm, and in areas of greater disorganization of myocyte bundles, usually surrounding blood vessels or near endocardium. Myocyte staining appeared to be perinuclear and cytoplasmic. PECAM-1 staining was detected at varying intensity in myocytes from both right and left ventricles in all 21 HLHS hearts examined. Immunohistochemical staining of four of these patients (Panels C – F), as well as from one pediatric nonsuitable donor (Panel A) and one retransplanted for rejection (Panel B) are shown in Fig. 4. Immunohistochemical detection of PECAM-1 was performed in many other heart sections from patients without the diagnosis of HLHS, including nonsuitable pediatric
Fig. 4. PECAM staining of pediatric heart tissue. (A) and (B) demonstrate staining of endothelium only in non-HLHS pediatric heart tissue. (A) is from an 18-month-old nonsuitable donor, (B) is from a 9-year-old retransplanted for rejection (PECAM-1 immunostain, original magnification 150). (C) – (F) show PECAM staining in myocytes as well as endothelium in hearts from four HLHS patients, ages 1 month, two at 2 months and 10 months. (C) demonstrates intense endothelial and myocyte staining (PECAM-1 immunostain, original magnification 150). (D) with myocyte staining around ‘‘sinusoids’’ (PECAM-1 immunostain, original magnification 25). (E) shows intense staining of disorganized myocytes (PECAM-1 immunostain, original magnification 150). (F) demonstrates similar intensity of staining in both myocytes and endothelial cells (PECAM-1 immunostain, original magnification 150).
donors (n = 2), pediatric transposition of the great vessels (n = 1), pediatric mitral stenosis (n = 1), pediatric IDC (n = 2), pediatric transplant rejection (n = 1), pediatric myocarditis (n = 1), pediatric hypoplastic right heart (n = 2), adult patients transplanted for heart failure (n = 4), including an 18-year-old male with IDC, and adult nonfailing, nonsuitable donors (n = 2). All samples showed strong endothelial-cell staining without evidence of the myocyte PECAM-1 expression seen in HLHS (data not shown). To demonstrate that myocyte PECAM-1 expression is not associated with myocyte disarray, we also examined right and left ventricles from three transgenic mice expressing a MHC mutation [32] (tissue kindly provided by Leslie A. Leinwand). The ventricles showed the disordered myocyte pattern typical of hypertrophic cardiomyopathy and intense PECAM-1 staining of endothelial cells, but no staining of myocytes.
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Fig. 5. Western blot analysis of PECAM-1 expression. Total protein was harvested from the left ventricle of three HLHS hearts and two normal pediatric donors. A total of 30 mg of protein was separated on a 7.5% polyacrylamide gel, transferred to nitrocellulose membranes and probed with a 1:1000 dilution of anti-PECAM-1 antibody and detected using ECL reagents. Densitometry was performed using a Sun Microsystems workstation.
3.5. PECAM-1 Western blotting Western blot experiments have been performed to characterize PECAM-1 protein expression in human heart tissue from HLHS patients and unaffected individuals. Total protein was isolated from the right and left ventricles, separated on a 7.5% polyacrylamide gel, transferred to nitrocellulose and probed with anti-PECAM-1 monoclonal antibody. Two antibodies from different suppliers (R&D Systems and DAKO) were used and both antibodies detected a single protein band of 130 kDa corresponding to the predicted size of the full-length PECAM-1. The data obtained using the DAKO antibody is shown in Fig. 5. Densitometry analysis of ventricular samples from three HLHS patients and two normal left ventricles suggest that the concentration of PECAM-1 is elevated in the HLHS samples (average integrated density of 5.52 vs. 1.55, Fig. 5).
4. Discussion HLHS accounts for approximately 7 –8% of heart disease producing symptoms in the first year of life and is the most common cause of death from heart disease in the first week after birth [3]. While there is a considerable body of literature on the diagnosis and surgical treatment of HLHS, there is no information on gene expression in HLHS ventricles. Histologic examination of tissue sections from LVs and RVs from patients with HLHS shows overall disorganization of both muscular and vascular elements, while individual myocytes and endothelial cells show histologic evidence of differentiation. Immunohistochemical detection of N-cadherin clearly shows the presence of intercalated discs. With quantitative PCR, we have shown that while left and right ventricular tissues from HLHS hearts express the fetal or ‘‘heart failure’’ gene expression
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pattern, the ventricular myocytes are clearly differentiated. Analysis of protein expression by 2-D gel electrophoresis and MALDI-TOF MS also confirm that HLHS left ventricular myocytes are differentiated. Using immunohistochemistry, we have found that HLHS myocytes in all patient samples are inappropriately expressing PECAM-1. Ectopic expression of PECAM-1 was specific to myocytes from HLHS patients and was absent from all adult and pediatric ventricular samples as well as from myocytes from transgenic mice with hypertrophic cardiomyopathy. The specificity of the anti-PECAM-1 monoclonal antibody used in the immunohistochemistry experiments and a second antibody from a second supplier demonstrates that the same protein species is being recognized in both the HLHS and unaffected tissue samples. Western blot analysis although not quantitative does suggest that PECAM-1 protein expression is elevated in HLHS ventricles as compared to normal. PECAM-1 is a member of the CAM class of proteins that are expressed as membrane spanning proteins and has a primary role in regulating tissue morphogenesis. PECAM-1 is an adhesion receptor belonging to the immunoglobulin superfamily and functions in cell – cell recognition or adhesion. PECAM-1 functions as an endothelial cell – CAM. CAMs such as PECAM-1 have a primary role in tissue morphogenic regulation and the normal physiological role of PECAM-1 is to regulate endothelial cell – cell interactions and endothelial cell binding to the basement membrane. It is also found on platelets and some white blood cells, where it is involved in transendothelial migration. Protein expression studies have demonstrated that PECAM-1 is leukocyte and endothelial cell-specific, and that PECAM-1 is not expressed in cardiac myocytes at any time during development or in any other cardiac disease [20]. In addition, studies using PECAM-1 null mice have clearly demonstrated that PECAM-1 expression is not necessary for normal cardiovascular development in the mouse [24], as these mice have no discernible cardiovascular malformations. Devaux et al. [33] have previously shown that PECAM-1 is not expressed in myocytes from normal and failing human hearts. PECAM-1 mRNA has been shown to be expressed in fetal human myocardium although the cell type responsible or that PECAM-1 protein is expressed has not been confirmed [23]. The fact that PECAM-1 is not required for normal cardiovascular development offers no guidance as to the possible consequences of ectopic expression of this protein in myocytes. In fact, it is very likely that inappropriate expression of a functionally active and appropriately membrane-localized CAM would significantly affect tissue morphogenesis. However, it is currently unclear if HLHS syndrome is the result of a single or multiple developmental abnormalities. It is possible that another gene under the same regulatory control as PECAM-1 could be responsible for HLHS. Alternatively, ectopic PECAM-1 expression could be an effect of some upstream developmental program
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error that results in HLHS. The observations that HLHS is characterized histologically by focal areas of disorganized bundles of small myocytes intermingled with small, thinwalled, haphazardly arranged blood vessels and large, thin-walled ‘‘sinusoids’’ and that PECAM-1 is the only protein identified to date as being inappropriately expressed in HLHS cardiac myocytes indicate that inappropriate expression of PECAM-1 or a related gene under the same regulatory control may be responsible for the disorganization of HLHS cardiac myocytes and potentially the higher order cardiac structural abnormalities associated with this disease.
[9]
[10]
[11]
[12]
[13]
5. Summary Immunohistochemical analysis of pediatric heart tissue from patients with HLHS revealed PECAM staining in myocytes and endothelium. PECAM is normally found only in endothelium and some white blood cells. Differentiation of myocytes from HLHS patients was demonstrated by identification of striations and intercalated discs, as well as by expression of genes found in differentiated heart tissue.
[14]
[15]
[16]
Acknowledgments The author gratefully acknowledge the technical assistance provided by the University of Colorado Health Sciences Center, Biochemical Mass Spectroscopy Facility, Mark W. Duncan, PhD, Director.
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T.J. Bohlmeyer et al. / Cardiovascular Pathology 12 (2003) 23–31 [25] Feldman AM, Ray PE, Bristow MR. Expression of alpha-subunits of G proteins in failing human heart: a reappraisal utilizing quantitative polymerase chain reaction. J Mol Cell Cardiol 1991;23:1355 – 8. [26] Lowes BD, Minobe W, Abraham WT, Rizeq MN, Bohlmeyer TJ, Quaife RA, Roden RL, Dutcher DL, Robertson AD, Voelkel NF, Badesch DB, Groves BM, Gilbert EM, Bristow MR. Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium. J Clin Invest 1997;100:2315 – 24. [27] Wessel D, Flugge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 1984;138:141 – 3. [28] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 – 54. [29] Clauser KR, Hall SC, Smith DM, Webb JW, Andrews LE, Tran HM, Epstein LB, Burlingame AL. Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma
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Hypoplastic left heart syndrome myocytes are differentiated but possess a unique phenotype Teresa J. Bohlmeyer, Steve Helmke, Shuping Ge, Jennifer Lynch, Gary Brodsky, James H. Sederberg II, Alastair D. Robertson, Wayne Minobe, Michael R. Bristow, M. Benjamin Perryman* Department of Medicine, Division of Cardiology, University of Colorado Health Sciences Center, Denver, CO, USA Department of Pediatrics (SG), University of Colorado Health Sciences Center, Denver, CO, USA Received 5 February 2002; received in revised form 15 July 2002; accepted 22 July 2002
Abstract Introduction: Hypoplastic left heart syndrome (HLHS) is the term used to describe a group of congenital malformations characterized by marked underdevelopment of the left side of the heart. HLHS accounts for nearly 25% of cardiac deaths in the first year of life. Although much has been reported regarding diagnosis, gross morphology and surgical treatment, no information on gene expression in HLHS myocytes is available. Methods: We examined heart tissue from patients with HLHS using routine histology, immunohistochemistry, quantitative polymerase chain reaction (PCR), two-dimensional (2-D) gel electrophoresis and protein identification by mass spectrometry. Results: Histologic examination of right and left ventricles from HLHS patients revealed characteristic features of myocyte differentiation, including striations and intercalated disc formation. Immunohistochemical staining using antibody to N-cadherin demonstrated clear development of intercalated discs between myocytes. However, many of the myocytes contained scant cytoplasm and were grouped in small, disorganized bundles separated by abundant connective tissue and dilated, thin-walled vessels. Quantitative PCR analysis demonstrated that both left and right ventricular tissue from HLHS hearts expressed the fetal or ‘‘heart failure’’ gene expression pattern. Two-dimensional gel electrophoresis and protein identification by mass spectrometry also confirmed that myocytes from HLHS ventricles were differentiated but expressed the fetal isoform of some cardiac specific proteins. However, HLHS myocytes in all of the heart samples (n = 21) were inappropriately expressing platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), a member of the cell adhesion molecule (CAM) family that has a primary role in the regulation of tissue morphogenesis. These findings indicate that myocytes from HLHS syndrome patients, while differentiated, have a unique gene expression pattern. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Hypoplastic left heart syndrome; Myocytes; Gene expression; Immunohistochemistry; Mass spectrometry
1. Introduction The term hypoplastic left heart syndrome (HLHS) is used to describe a group of malformations characterized by marked underdevelopment of the left side of the heart. The anatomic abnormalities include underdevelopment of the left atrium and ventricle, stenosis or atresia of the aortic or mitral orifices, and marked hypoplasia of the ascending aorta [1,2]. HLHS accounts for approximately 7– 8% of heart disease producing symptoms in the first year of life * Correspondence author. Department of Medicine, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B139, Denver, CO 80246, USA. Tel.: +1-303-315-3259. E-mail address: [email protected] (M.B. Perryman).
and is the most common cause of death from heart disease in the first week after birth [3]. It accounts for nearly 25% of cardiac deaths in the first year of life (New England Regional Infant Cardiac Registry). The prognosis for most patients with HLHS is bleak and treatment is surgery to repair the defects or, more rarely, cardiac transplantation. HLHS is unlike most other complex congenital heart diseases in that abnormalities in other organ systems are rare. Thus, children with HLHS are, except for heart abnormalities, normal [4]. The genetic basis of HLHS is not clear, although in some cases the syndrome has been reported to be associated with chromosomal abnormalities [5,6]. Microscopic examination of tissue sections from both right and left ventricles of patients with HLHS shows disorganized, variably sized bundles of myocytes coursing
1054-8807/02/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 4 - 8 8 0 7 ( 0 2 ) 0 0 1 2 7 - 8
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through abundant loose connective tissue and scattered, dilated, thin-walled blood vessels. Myocytes are focally small with scant cytoplasm. It is not known if HLHS myocytes express proteins typical of differentiated myocytes or remain significantly undifferentiated. Platelet-endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily [7]. The 130-kDa fulllength protein possesses six extracellular immunoglobulin domains, a transmembrane portion and a cytoplasmic domain [8]. Functionally, PECAM-1 is an adhesion molecule with both homophilic and heterophilic binding. The homotypic binding is important in leukocyte transendothelial migration [9]. The heterotypic ligands have been reported to include integrin, avb3 [10] and glycosaminoglycans [11]. PECAM-1 is present on the surface of platelets, some white blood cells and endothelial cells [7,12], and is strongly expressed by all endothelial cells and to a lesser extent on several types of leukocytes [13]. Immunohistochemical staining for PECAM-1 is routinely used to demonstrate angiogenesis in several types of cancer [14 –19]. Protein expression studies have demonstrated that PECAM-1 is leukocyte and endothelial cell-specific and that PECAM-1 is not expressed in mouse cardiac myocytes at any time during development or in any cardiac diseases [20 – 22]. However, Hwang et al. reported expression of PECAM-1 in 8– 12-week fetal human hearts but not in older fetal or adult hearts. Since this analysis was cDNA-based, it cannot be determined if PECAM-1 mRNA expression in fetal hearts of this age is localized to myocytes or other cell types within the myocardium [23]. In addition, PECAM-1 null mice have no discernible cardiovascular malformations and therefore PECAM-1 expression is not necessary for normal cardiovascular development in the mouse [24]. With improvements in early diagnosis of HLHS and the increasing potential for surgical intervention in the fetus, it is essential to determine the pathophysiological basis for the ventricular malformations and to determine the potential for HLHS myocytes to respond to normalized ventricular flow. If the ventricular myocytes are unable to respond appropriately following surgical correction, early intervention may be futile. We have used immunohistochemistry, quantitative reverse transcription polymerase chain reaction (QRTPCR) and two-dimensional (2-D) gel electrophoresis coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to determine that HLHS left ventricular myocytes have a differentiated but unique phenotype. The myocytes express mRNAs and proteins characteristic of differentiated, failing cardiac myocytes and form striations and intercalated discs. However, HLHS myocytes in all patient samples (n = 21) inappropriately express PECAM-1, a member of the CAM family that has a primary role in the regulation of tissue morphogenesis. Cardiac myocytes have never been shown to express PECAM-1 at any stage of development or in any disease state. These findings indicate that myocytes from HLHS
syndrome patients, while differentiated, have a unique gene expression pattern.
2. Methods 2.1. Tissue procurement Explanted human heart tissue was obtained during heart transplantation from pediatric HLHS patients (n = 21, ages 6 days to 10 months), nonsuitable pediatric donors (n = 2, ages 18 months and 8 years), pediatric transposition of the great vessels (n = 1, age 20 years), mitral stenosis (n = 1, age 4 years), idiopathic dilated cardiomyopathy (IDC; n = 2, ages 1 and 10 months), transplant rejection (n = 1, age 9 years), myocarditis (n = 1, age 2 months), hypoplastic right heart (n = 2, ages 2 and 5 months), adult patients transplanted for heart failure (n = 4, ages 18 – 64 years), including an 18-year-old male with IDC, and adult nonfailing, nonsuitable donors (n = 2, ages 28 and 45 years). Tissue was taken from both the right and left ventricular free wall, when available. All patients had been taking a variety of drugs, primarily diuretics, immunosupressants and positive inotropes. 2.2. Histology Heart tissue was formalin-fixed, routinely processed, paraffin-embedded, cut to 5-mm-thick sections onto Fisherbrand superfrost/Plus slides, and stained with hematoxylin and eosin and trichrome. 2.3. Immunohistochemistry Immunohistochemical detection was performed using the labeled avidin –biotin (LAB) system (DAKO LSAB + kit, peroxidase), monoclonal mouse antihuman endothelial cell, CD31 (PECAM-1) antibody (DAKO) and polyclonal rabbit antimouse, rat, human N-cadherin (Santa Cruz Biotechnology). Briefly, 5-mm-thick sections of formalin-fixed, paraffin-embedded heart tissue were deparaffinized in xylene, rehydrated and microwaved in 10-mM citrate buffer for antigen retrieval. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide. Appropriately diluted primary antibody was applied, followed by the biotinylated universal (swine antigoat, mouse, rabbit) secondary antibody and finally peroxidase-labeled streptavidin, which binds to biotin. Appropriate blocking steps using dilute bovine serum albumin, nonimmune swine serum and biotin – avidin were performed. The color reaction was developed using DAB (3,30-diaminobenzidine), resulting in a reddish-brown precipitate at the antigen site. Sections were counterstained with hematoxylin. Negative controls using appropriately diluted nonimmune serum instead of primary antibody were performed for each experiment. Endothelial cells lining blood vessels in each tissue section served as positive controls.
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2.4. Quantitative PCR Using a quantitative PCR technology developed within our group [25,26], we examined the expression of several fetal genes, b1- and b2-adrenergic receptors (AR), a- and b-myosin heavy chain (MHC), atrial natriuetic peptide (ANP) and sarcoplasmic reticulum calcium ATPase (SRCA), and compared their expression to that of adult nonfailing myocardium. The detailed methodology and values for the adult heart measurements have been previously reported [26]. 2.5. 2-D gel electrophoresis Approximately 50 mg of ventricular tissue was homogenized on ice using a ground glass homogenizer in 500 ml of sample buffer I from Genomic Solutions (GSI), 50 mM Tris, pH 8.0, 0.2 M DTT, 0.3% SDS supplemented with a protease inhibitor cocktail of 1 mM AEBSF, 0.1 mM leupeptin and 10 mg/ml aprotinin, all from Sigma. A 50-ml aliquot of sample buffer II (GSI; 0.5 M Tris, pH 8.0, 50 mM MgCl2, 2000 units/ml DNAse I, 750 units/ml RNAse A) was added and the mixture incubated on ice for 10 min to digest nucleic acids. The proteins were quantitatively precipitated by an adaptation of the method of Wessel and Flugge [27]. To the approximately 600 ml of homogenate was added methanol:chloroform (4:1, 750 ml) and the mixture was vortexed vigorously at room temperature. (All subsequent steps were performed at room temperature.) The mixture was centrifuged at 16,000 g for 5 min to separate phases, and the upper aqueous phase was carefully removed so as not to disturb the protein disc at the interphase. Methanol (450 ml) was added, and the sample thoroughly mixed to give a single phase. The protein was pelleted by centrifugation (16,000 g, 5 min) and the supernatant removed. The protein pellet was then dried in a Speedvac for 30 min. IEF sample buffer (500 ml) was added to each pellet; the samples were incubated overnight at room temperature. IEF sample buffer was prepared from IPG strip rehydration buffer (GSI), 8 M urea, 2% CHAPS, 2% ampholytes by supplementing it with 2 M thiourea and 50 mM DTT. After incubation, vigorous mixing and homogenization with a pellet pestle (Fisher) dispersed any undissolved material. Centrifugation (16,000 g, 10 min) pelleted unsolubilized material, mainly connective tissue. The supernatant was collected and a 20-ml aliquot was diluted 1– 50 with water and assayed for protein by the method of Bradford [28] (BioRad Protein Assay). IPG strips (18 cm, nonlinear pH 3– 10, GSI) were rehydrated with 2.5-mg protein in 400 ml of IEF sample buffer. After overnight rehydration in a humidified chamber, the IPG strips were subjected to 100,000 V h of focusing over 24 h. The strips were equilibrated for 10 min in equilibration buffer I (GSI) and for 10 min in equilibration buffer II (GSI) to reduce and alkylate the proteins with iodoacetamide. The strips were then placed on precast 10% polyacrylamide tricine double gels (GSI) and electrophoresed at 14 W/gel
25
for 6 h. The gels were stained with colloidal Coomassie (Invitrogen) and destained with water. Images of the gels were captured with a Umax scanner. 2.6. In-gel tryptic protein digest Protein spots were excised from the gels with a truncated 1-ml pipet tip. Gel pieces were washed 2 for 10 min with 100 ml 50% acetonitrile (CH3CN)/25 mM ammonium bicarbonate (NH4HCO3) and then once with 100 ml 100% CH3CN. The gel pieces were dried in a Speedvac for 15 min. Promega sequencing grade trypsin (20 ml of 20 mg/ml in 10 mM NH4HCO3) was added and the gel pieces were allowed to rehydrate on ice for 20 min, incubated overnight at 37. The next day the peptides were extracted into 50 ml of 50% CH3CN/5% trifluoroacetic acid (TFA) with vigorous mixing (1 h). The extracts were transferred to fresh tubes and taken to dryness (Speedvac). The peptides were then redissolved in 0.5% TFA (20 ml). ZipTips (C18, 0.6 ml bed volume, Millipore) were wetted with 50% CH3CN and equilibrated in 0.1% TFA. Peptides were bound by pipetting 10 through the bed. The tips were then washed 2 with 0.1% TFA. Peptides were eluted with 50% CH3CN/0.1% TFA (5 ml) by pipetting 5 through the bed. 2.7. Mass spectrometry and protein identification The peptide eluate (0.5 ml) and matrix solution (0.5 ml) were thoroughly mixed on a clean stainless steel MALDITOF MS target and allowed to dry. The matrix solution was 10 mg/ml a-cyano-4-hydroxycinnamic acid dissolved in 50% CH3CN/0.1% TFA. A standard mix of bradykinin, angiotensin, glu-fibrino-peptide and ACTH 18 – 30 in matrix was also applied to an adjacent region of the target. MALDI-TOF spectra were acquired on a Voyager-De Pro (PerSeptive Biosystems) instrument operating in reflector mode. Following initial data collection using external calibration on the standard mix, the spectra of unknowns were recalibrated using autolytic tryptic fragments as internal mass markers. Mono-isotopic peptide masses with a signal to noise of greater than 5:1 were entered into the Protein Prospector MS-Fit program (www.prospector.ucsf.edu). This program finds the best fit of observed tryptic peptides to predicted tryptic peptides from known proteins [29]. These masses were searched at a tolerance of 0.1 Da against the SwissProt database. Cysteine carbamidomethylation was permitted as a modification to account for alkylation by iodoacetamide. Oxidation of methionine was also permitted because this has been observed after colloidal Coomassie staining. In the case of highly conserved proteins such as actin the search was limited to Homo sapiens. Our criteria for the positive identification of a protein included that it be the top MSfit candidate, that its MOWSE score be severalfold higher than the next best candidate, that the majority of peptides matched this protein and that at least six peptides matched this protein. The predicted molecular weight (MW)
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and pI were also considered in making the identification (Table 2). 2.8. Western blotting Total protein (30 mg) was separated on a 7.5% polyacrylamide gel, transferred to nitrocellulose membrane, probed with a 1:1000 dilution of anti-PECAM-1 antibody and detected using enhanced chemiluminescence (ECL) (Renaissance NEN). The image of the Western blot was captured and densitometry analysis performed using a SUN workstation.
3. Results 3.1. Histopathology Hematoxylin and eosin and trichrome-stained sections of HLHS ventricles revealed similar histologic features in both right and left ventricles. All sections contained large areas of randomly oriented, disorganized bundles of myocytes, with variability of myocyte size found between bundles. Some bundles were composed of normally proportioned and sized myocytes with centrally located, ovoid nuclei; others contained crowded, thin myocytes with scant cytoplasm and an increased nuclear to cytoplasmic ratio. Abundant loose connective tissue was found separating myocyte bundles and, focally, surrounding individual myocytes. Large, thinwalled ‘‘sinusoids’’ were observed scattered throughout the tissue, surrounded by more dense fibrous tissue. Even in areas containing normal-sized myocytes with only thin fibrous bands separating myocyte bundles, the individual myocytes appeared randomly oriented and disorganized. In some patients, the histologically abnormal areas were greater in the right ventricle than in the left. Many of these
Fig. 2. N-Cadherin immunostaining. Immunohistochemical demonstration of intercalated discs by detection of N-cadherin. Striations are also evident (LV from a different 3-month-old HLHS patient than shown in Fig. 1, N-cadherin immunostain, original magnification 150).
features can be seen in the trichrome-stained HLHS left ventricular section shown in Fig. 1. 3.2. N-Cadherin expression Immunohistochemical detection of N-cadherin revealed clear development of intercalated discs between myocytes even in areas of marked disorganization and abnormal myocyte morphology. Striations are also clearly seen (Fig. 2). 3.3. mRNA and protein expression One of the characteristics of failing myocardium is the reactivation of expression of a number of fetal genes coupled with down-regulation of a number of genes encoding contractile function regulating proteins in the adult ventricle. Using quantitative PCR technology, we examined left ventricular tissues from five HLHS patients and two normal pediatric donor hearts for expression of some of these genes. The mean values for HPLS measurements reported as mRNA molecules 105/mg of total mRNA is shown in Table 1. The P-values and ratios for all measurements are also shown. HLHS left ventricle mRNA expression of b1-AR, a-MHC, b-MHC and SERCA mRNA concentrations are reduced when compared to the normal left ventricles. The mRNA concentrations for ANF and Table 1 mRNA abundance in HLHS ventricles
Fig. 1. Hypoplastic left heart syndrome. Disorganized bundles of myocytes, large areas of fibrosis and abnormal vascular formations characterize both left and right ventricles in patients with HLHS (LV from 3-month-old HLHS patient, trichrome stain, original magnification 25).
mRNA
HLHS LV mean
Donor LV mean
P-value
Ratio
b1AR b2AR aMHC bMHC ANP SERCA
0.28 2.40 1.88 36.59 27.97 75.23
0.45 2.36 17.70 59.41 27.89 206.47
.33 1.00 .08 .18 1.00 .08
0.62 1.02 0.11 0.62 1.00 0.36
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27
Fig. 3. Colloidal Coomassie stained 2-D gels of adult (IDC) and pediatric (HLHS) human heart. Left ventricular tissue was prepared as described in Section 2 and the proteins resolved by 2-D gel electrophoresis. Proteins are identified by peptide mass mapping as described. Note the similarity in pattern between the pediatric and adult heart proteins. The atrial light chain 1 is an embryonic form expressed in the pediatric ventricle that disappears with maturity, but is reexpressed at a low level in failing adult human heart.
b2-AR are essentially unchanged as compared to normal controls. These data indicate that the HLHS left ventricles express at least a portion of the heart failure or fetal gene expression pattern. Even more importantly, these data clearly indicate that the HLHS ventricles are comprised of differentiated myocytes expressing the cardiac myocyte specific genes, SERCA 2a, a-MHC and ANF. Since mRNA concentrations do not necessarily correlate with the concentration of the corresponding protein we used 2-D gel electrophoresis coupled with protein identification by MALDI-TOF MS to compare HLHS ventricular protein expression to normal and failing ventricular protein expression. The protein expression profile of HLHS pediatric LV was compared to that of failing (IDC) adult LV using highresolution large format 2-D gel electrophoresis (Fig. 3). The protein spot pattern of the HLHS pediatric LV was very similar to that of the adult LV. While there were some differences in the minor spots, all the major spots were in the same position and had the same intensity on both gels,
with one exception, the expression of ALC-1 in the HLHS but not in the normal left ventricle. This indicates that the protein expression of the HLHS pediatric LV is very similar to the adult LV and that the HLHS ventricle is composed of differentiated myocytes. In order to identify the major protein spots and to confirm that the same proteins were expressed in both HLHS pediatric LV and adult LV, the spots were excised from both gels, digested with trypsin and analyzed by MALDI-TOF MS. Spectra were acquired in the highresolution reflector mode and the autolytic tryptic fragments were used as internal mass markers to increase mass accuracy. For each peptide, the mono-isotopic mass was identified and recorded for database searches. The SwissProt database was searched at a tolerance of 0.1 Da using Protein Prospector MS-Fit. Protein identification required a high MOWSE score relative to other candidates, matching a majority of the peptides, matching at least six peptides and an experimentally determined MW and pI consistent
Table 2 Proteins identified by peptide mass matching, MW and pI Protein
Theoretical MW
Experimentally determined MW
Theoretical pI
Experimentally determined pI
Peptides matched/submitted
% AA coverage
Tropomyosin ALC-1 Actin Troponin T M-CK VLC-2 VLC-1 Fatty acid binding-protein a-Crystallin B-chain Myoglobin
32,709 21,565 42,019 34,591 43,102 18,790 21,932 14,858 20,159 17,184
33,000 25,000 42,000 40,000 43,000 19,000 22,000 15,000 21,000 17,000
4.69 4.97 5.23 5.13 6.77 4.92 5.03 6.29 6.76 7.14
4.7 5.4 5.6 5.6 6.4 5.3 5.5 6.2 6.6 7.1
11/12 9/9 10/15 9/9 10/12 11/12 9/11 5/10 7/13 6/7
30 46 30 32 29 54 44 35 32 44
Following in-gel digestion with trypsin, proteins are identified by peptide mass matching. Identifications are confirmed by comparing the theoretical MWs and pI’s of these proteins with the MWs and pI’s experimentally determined from their position on the 2-D gels. The number of peptides matched to the predicted protein over the number of peptides submitted to MSfit is shown. The percent coverage is the number of amino acids in the matched peptides as a percentage of the total amino acids in the predicted protein.
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with the predicted values (Table 2). The experimentally determined MWs matched very closely to the predicted MWs except for atrial light chain-1 (ALC-1) and troponin T, which had slightly lower mobilities than expected. The experimentally determined pI’s were all within 0.5 pH units of the predicted values. (Actual pI’s are modified by charge distributions and interactions, and will often vary from theoretical pI’s, which are estimated from amino acid composition.) ALC-1 is calculated to have almost the same MW and pI as VLC-1, but appears somewhat larger and more acidic than VLC-1. This has been observed in other 2-D gel studies of human heart [30,31]. ALC-1 is significantly less abundant in the adult heart and is the embryonic isoform of LC-1 in the ventricle. During development, there is a switch in the ventricle from ALC-1 to the adult isoform VLC-1. In the pediatric heart, this switch is about half complete. The adult ventricle expresses almost exclusively VLC-1; but, in heart failure, it has been reported that there is re-expression of the embryonic isoform, ALC-1 [30,31]. The adult heart shown in Fig. 3 is a failing heart from a patient with IDC and this expression of ALC-1 probably reflects the fetal gene program. The major spots identified in Fig. 3 represent some of the most abundant proteins of the heart. As expected these include the contractile apparatus proteins actin, tropomyosin, myosin light chains and troponin T. Major metabolic proteins include myoglobin, fatty acid binding protein and M-CK. The most abundant member of the small heat shock family protein in heart is a-crystallin B-chain is also present. Analysis of the protein expression pattern of demonstrates that the myocytes of the HLHS ventricle are in a differentiation state similar to adult ventricular myocytes. 3.4. PECAM-1 expression Examination of sections of right and left ventricle used for immunohistochemical detection of PECAM-1 from 21 children transplanted for HLHS showed a distinct brown staining of endothelium lining both the large and small blood vessels. In addition, patchy and, in some patients, extensive areas of all sections examined contained distinct brown staining within myocytes, particularly in areas in which the myocytes appeared smaller, with less cytoplasm, and in areas of greater disorganization of myocyte bundles, usually surrounding blood vessels or near endocardium. Myocyte staining appeared to be perinuclear and cytoplasmic. PECAM-1 staining was detected at varying intensity in myocytes from both right and left ventricles in all 21 HLHS hearts examined. Immunohistochemical staining of four of these patients (Panels C – F), as well as from one pediatric nonsuitable donor (Panel A) and one retransplanted for rejection (Panel B) are shown in Fig. 4. Immunohistochemical detection of PECAM-1 was performed in many other heart sections from patients without the diagnosis of HLHS, including nonsuitable pediatric
Fig. 4. PECAM staining of pediatric heart tissue. (A) and (B) demonstrate staining of endothelium only in non-HLHS pediatric heart tissue. (A) is from an 18-month-old nonsuitable donor, (B) is from a 9-year-old retransplanted for rejection (PECAM-1 immunostain, original magnification 150). (C) – (F) show PECAM staining in myocytes as well as endothelium in hearts from four HLHS patients, ages 1 month, two at 2 months and 10 months. (C) demonstrates intense endothelial and myocyte staining (PECAM-1 immunostain, original magnification 150). (D) with myocyte staining around ‘‘sinusoids’’ (PECAM-1 immunostain, original magnification 25). (E) shows intense staining of disorganized myocytes (PECAM-1 immunostain, original magnification 150). (F) demonstrates similar intensity of staining in both myocytes and endothelial cells (PECAM-1 immunostain, original magnification 150).
donors (n = 2), pediatric transposition of the great vessels (n = 1), pediatric mitral stenosis (n = 1), pediatric IDC (n = 2), pediatric transplant rejection (n = 1), pediatric myocarditis (n = 1), pediatric hypoplastic right heart (n = 2), adult patients transplanted for heart failure (n = 4), including an 18-year-old male with IDC, and adult nonfailing, nonsuitable donors (n = 2). All samples showed strong endothelial-cell staining without evidence of the myocyte PECAM-1 expression seen in HLHS (data not shown). To demonstrate that myocyte PECAM-1 expression is not associated with myocyte disarray, we also examined right and left ventricles from three transgenic mice expressing a MHC mutation [32] (tissue kindly provided by Leslie A. Leinwand). The ventricles showed the disordered myocyte pattern typical of hypertrophic cardiomyopathy and intense PECAM-1 staining of endothelial cells, but no staining of myocytes.
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Fig. 5. Western blot analysis of PECAM-1 expression. Total protein was harvested from the left ventricle of three HLHS hearts and two normal pediatric donors. A total of 30 mg of protein was separated on a 7.5% polyacrylamide gel, transferred to nitrocellulose membranes and probed with a 1:1000 dilution of anti-PECAM-1 antibody and detected using ECL reagents. Densitometry was performed using a Sun Microsystems workstation.
3.5. PECAM-1 Western blotting Western blot experiments have been performed to characterize PECAM-1 protein expression in human heart tissue from HLHS patients and unaffected individuals. Total protein was isolated from the right and left ventricles, separated on a 7.5% polyacrylamide gel, transferred to nitrocellulose and probed with anti-PECAM-1 monoclonal antibody. Two antibodies from different suppliers (R&D Systems and DAKO) were used and both antibodies detected a single protein band of 130 kDa corresponding to the predicted size of the full-length PECAM-1. The data obtained using the DAKO antibody is shown in Fig. 5. Densitometry analysis of ventricular samples from three HLHS patients and two normal left ventricles suggest that the concentration of PECAM-1 is elevated in the HLHS samples (average integrated density of 5.52 vs. 1.55, Fig. 5).
4. Discussion HLHS accounts for approximately 7 –8% of heart disease producing symptoms in the first year of life and is the most common cause of death from heart disease in the first week after birth [3]. While there is a considerable body of literature on the diagnosis and surgical treatment of HLHS, there is no information on gene expression in HLHS ventricles. Histologic examination of tissue sections from LVs and RVs from patients with HLHS shows overall disorganization of both muscular and vascular elements, while individual myocytes and endothelial cells show histologic evidence of differentiation. Immunohistochemical detection of N-cadherin clearly shows the presence of intercalated discs. With quantitative PCR, we have shown that while left and right ventricular tissues from HLHS hearts express the fetal or ‘‘heart failure’’ gene expression
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pattern, the ventricular myocytes are clearly differentiated. Analysis of protein expression by 2-D gel electrophoresis and MALDI-TOF MS also confirm that HLHS left ventricular myocytes are differentiated. Using immunohistochemistry, we have found that HLHS myocytes in all patient samples are inappropriately expressing PECAM-1. Ectopic expression of PECAM-1 was specific to myocytes from HLHS patients and was absent from all adult and pediatric ventricular samples as well as from myocytes from transgenic mice with hypertrophic cardiomyopathy. The specificity of the anti-PECAM-1 monoclonal antibody used in the immunohistochemistry experiments and a second antibody from a second supplier demonstrates that the same protein species is being recognized in both the HLHS and unaffected tissue samples. Western blot analysis although not quantitative does suggest that PECAM-1 protein expression is elevated in HLHS ventricles as compared to normal. PECAM-1 is a member of the CAM class of proteins that are expressed as membrane spanning proteins and has a primary role in regulating tissue morphogenesis. PECAM-1 is an adhesion receptor belonging to the immunoglobulin superfamily and functions in cell – cell recognition or adhesion. PECAM-1 functions as an endothelial cell – CAM. CAMs such as PECAM-1 have a primary role in tissue morphogenic regulation and the normal physiological role of PECAM-1 is to regulate endothelial cell – cell interactions and endothelial cell binding to the basement membrane. It is also found on platelets and some white blood cells, where it is involved in transendothelial migration. Protein expression studies have demonstrated that PECAM-1 is leukocyte and endothelial cell-specific, and that PECAM-1 is not expressed in cardiac myocytes at any time during development or in any other cardiac disease [20]. In addition, studies using PECAM-1 null mice have clearly demonstrated that PECAM-1 expression is not necessary for normal cardiovascular development in the mouse [24], as these mice have no discernible cardiovascular malformations. Devaux et al. [33] have previously shown that PECAM-1 is not expressed in myocytes from normal and failing human hearts. PECAM-1 mRNA has been shown to be expressed in fetal human myocardium although the cell type responsible or that PECAM-1 protein is expressed has not been confirmed [23]. The fact that PECAM-1 is not required for normal cardiovascular development offers no guidance as to the possible consequences of ectopic expression of this protein in myocytes. In fact, it is very likely that inappropriate expression of a functionally active and appropriately membrane-localized CAM would significantly affect tissue morphogenesis. However, it is currently unclear if HLHS syndrome is the result of a single or multiple developmental abnormalities. It is possible that another gene under the same regulatory control as PECAM-1 could be responsible for HLHS. Alternatively, ectopic PECAM-1 expression could be an effect of some upstream developmental program
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error that results in HLHS. The observations that HLHS is characterized histologically by focal areas of disorganized bundles of small myocytes intermingled with small, thinwalled, haphazardly arranged blood vessels and large, thin-walled ‘‘sinusoids’’ and that PECAM-1 is the only protein identified to date as being inappropriately expressed in HLHS cardiac myocytes indicate that inappropriate expression of PECAM-1 or a related gene under the same regulatory control may be responsible for the disorganization of HLHS cardiac myocytes and potentially the higher order cardiac structural abnormalities associated with this disease.
[9]
[10]
[11]
[12]
[13]
5. Summary Immunohistochemical analysis of pediatric heart tissue from patients with HLHS revealed PECAM staining in myocytes and endothelium. PECAM is normally found only in endothelium and some white blood cells. Differentiation of myocytes from HLHS patients was demonstrated by identification of striations and intercalated discs, as well as by expression of genes found in differentiated heart tissue.
[14]
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[16]
Acknowledgments The author gratefully acknowledge the technical assistance provided by the University of Colorado Health Sciences Center, Biochemical Mass Spectroscopy Facility, Mark W. Duncan, PhD, Director.
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