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Cardiovascular Research 2003 60(2):376-387; doi:10.1016/j.cardiores.2003.08.005
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Copyright © 2003, European Society of Cardiology

β-Catenin accumulates in intercalated disks of hypertrophic cardiomyopathic hearts

Laura Masuellia, Roberto Beib, Pamela Sacchettia, Ilaria Scappaticcia, Paola Francalancic, Loredana Albonicib, Anna Colettia, Camilla Palumbob, Marilena Minierid, Roberta Fiaccaventod, Felicia Carotenutod, Cristina Fantinid, Luciana Carosellae, Andrea Modestib and Paolo Di Nardo*,d

aDepartment of Experimental Medicine and Pathology, University of Rome "La Sapienza", Rome, Italy
bDepartment of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", Rome, Italy
cDepartment of Pathology, Bambino Gesu Children's Hospital-Research Institute Rome, Rome, Italy
dDepartment of Internal Medicine, Università di Roma Tor Vergata, Via Montpellier, 1, 00133 Rome, Italy
eDepartment of Internal Medicine and Geriatrics, Catholic University of Sacred Heart, Rome, Italy

*Corresponding author. Tel.: +39-6-72594215; fax: +39-6-2024130. Email address: dinardo{at}med.uniroma2.it

Received 3 April 2003; revised 6 August 2003; accepted 7 August 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: To evaluate whether cardiomyocyte membrane structure and cell/extracellular matrix adhesion alterations perturb the cadherin/catenin complex in the hypertrophic cardiomyopathy (HCM). Methods: Hypertrophic cardiomyopathic hamster (UM-X7.1 strain) and human hearts were studied by light and electron microscopy, Northern and Western blot analyses and immunohistochemistry. Results: Intercalated disks are disorganized in both hamster and human cardiomyopathic hearts; β-catenin is increased and accumulated in intercalated disks depriving cardiomyocyte nuclei of fundamental signals. The accumulation of β-catenin is post-translationally regulated by an increased Wnt expression, a simultaneous decrease in glycogen synthase kinase 3β (GSK3β) expression and a different expression pattern of adenomatous polyposis coli (APC) isoforms. Conclusion: The reorganization of cell/cell adhesion in cardiomyopathic hearts is mainly contributed by the cadherin/catenin system, which is differently regulated to sustain cell structural rather than signalling needs causing considerable consequences in the determination of cardiomyocyte phenotype and clinical outcome. The accumulation of β-catenin in intercalated disks could concur to increase myocardial wall stiffness and left ventricular end-diastolic pressure (LVEDP) in hypertrophic cardiomyopathic hamster and human hearts.

KEYWORDS Cardiomyopathy; Cell adhesion; Histopathology; Catenin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Advances in molecular genetics and biochemistry have allowed the identification of numerous mutations involved in the pathogenesis of cardiomyopathies. Nevertheless, the understanding of molecular pathways leading to different cardiomyopathic phenotypes remains rudimentary. Three different clinical phenotypes of cardiomyopathies are presently recognized: hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM). It has been hypothesized that a specific set of molecular pathways could account for each phenotype of the three major forms of cardiomyopathy. Early evidence suggested that sarcomeric mutations were responsible for HCM [1], while cytoskeletal mutations caused DCM [2]. Subsequently, it has been demonstrated that single mutations in sarcomeric genes can lead to a spectrum of cardiomyopathic phenotypes [3] indicating that the ultimate clinical outcome results from the integrated effects of multiple interacting factors (cardiomyopathic genotype, genetic background, hemodynamic stress, myocyte survival and subsequent fibrosis, calcium cycling, etc.). In order to unravel the complex interplay of different factors concurring to establish different cardiomyopathic phenotypes, genetically engineered mice were shown to be invaluable models. However, the spontaneously {delta}-sarcoglycan ({delta}-SG) null hamster also represents an intriguing experimental model of hereditary cardiomyopathy. In this animal model, the deletion of the promoter and first exon region of the above mentioned gene [4,5] can generate either the hypertrophic or dilated cardiomyopathic phenotype mimicking the human disease in which single mutations can result in different outcomes.

{delta}-SG is one of the four sarcoglycan subunits that, together with the β and {varepsilon} subunits and sarcospan, is normally expressed in cardiomyocytes and smooth muscle cells [6], while the expression of β and {varepsilon} subunits is restricted to skeletal and cardiac muscle [7,8]. Sarcoglycans combine with dystrophin, syntrophins, dystrobrevin, dystroglycans ({alpha} and {gamma} subunits) and sarcospan to constitute the dystrophin–glycoprotein complex (DGC), a multifunctional protein complex, connecting extracellular matrix with cytoskeleton and acting as transmembrane signaling complex [9]. The integrity of the complex is essential for the viability of muscle cells and its disruption caused by defects in dystrophin or one sarcoglycan subunit generates sarcolemmal instability, which, in turn, may make muscle fibers susceptible to cell damage and necrosis [10].

Recently, in engineered {delta}-SG deficient mice, the cardiomyopathy pathogenesis has been related to the lack of the sarcoglycan-sarcospan complex in vascular smooth muscle causing functional coronary alterations and widespread myocardial necrosis of ischemic origin [11,12]. However, the possibility that further unknown signaling pathways might be activated in {delta}-SG null cardiomyocytes cannot be excluded [13]. In fact, the perturbation of the sarcolemmal integrity caused by {delta}-SG ablation, very likely, implicates the activation of different signaling pathways as a consequence of plasmalemma structure and function alteration. Among others [14–16], adhesion structures, such as the cadherin/catenin complex, can suffer for the misalignment of the connection between extracellular ligands and cytoskeletal elements generating aberrant signals to the cell. Cadherins bind β- or {gamma}-catenin that, in turn, binds {alpha}-catenin. The latter binds cytoskeletal actin bridging extra- and intracellular milieu [17,18]. The integrity of the cadherin and catenin molecules is necessary for cell/cell adhesion. In addition to the mechanical function, the cadherin/catenin complex plays a role in signal transduction activating rapid and localized changes in adhesion to respond to extracellular signals [19]. In particular, β-catenin participates in adhesion processes when associated with cadherins, while it contributes to the Wnt/Wingless signaling cascade when freely present in the cytosol. [20–22]. Wnt signaling leads to stabilization of cytosolic β-catenin; in the absence of Wnt signals, β-catenin is phosphorylated by glycogen synthase kinase 3β (GSK3β), ubiquinated and degraded in proteasomes. Stabilized β-catenin enters the cell nucleus and associates with LEF/TCF transcription factor, which leads to the transcription of Wnt-target genes [23].

The present study was aimed at evaluating whether and to what extent the ablation of the {delta}-SG can perturb the cardiomyocyte membrane structure inducing alterations in the plasmalemma organization and membrane-related signaling pathways. In particular, the study has been focused on the analysis of the perturbation of the cadherin/catenin adhesion complex in the myocardium of cardiomyopathic hamsters and human beings.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Experimental model
Spontaneously {delta}-sarcoglycan-deficient hamsters (UM-X7.1 and TO2 strains) aged 30, 90 and 150 days were studied in comparison to age-matched healthy Golden Syrian (GSH) hamsters (Mesocricetus auratus). Animals were fed in our animal facility under the same standard conditions. The investigation conformed to the guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Human heart samples
Ventricular specimens of human myocardium were obtained from a 10-year-old boy submitted to partial left ventriculectomy (Batista procedure) [24] because of hypertrophic cardiomyopathy diagnosed by standard procedures [25]. Healthy human myocardium was obtained from a normal heart not implanted because of technical problems. The investigation conformed to the principles outlined in the Declaration of Helsinki.

2.3. Histopathology and ultrastructural analysis
Light microscopy and ultrastructural analyses were performed on myocardium fragments fixed in 2.5% glutaraldehyde in PBS pH 7.4 and processed for transmission electron microscopy as previously described [26]. For light microscopy, semithin sections were stained with tolouidine blue. Two independent observers evaluated three different hearts per each age group.

2.4. Western blot analysis
Electrophoresis of myocardium homogenates (100 µg protein/lane) from three different pools of hamster hearts (3 hearts/each) was carried out in denaturing 8% Tris–glycine polyacrylamide gels (SDS-PAGE) as previously described [27]. Filters were incubated with polyclonal antibodies against {alpha}-, β-, {gamma}-catenin, N-cadherin, adenomatous polyposis coli (APC; Santa Cruz, CA, USA), and then developed by a Supersignal West Pico chemiluminescence kit (Pierce, Rockford, IL, USA). Blots were simultaneously probed with anti-N-cadherin and {gamma}-catenin and, after stripping, with anti-{alpha}- and β-catenin antibodies. To ensure equal loading of proteins, Ponceau Red staining of membranes was performed each time after transfer and before immunoreaction. Since the staining intensity of an unknown protein migrating with an apparent molecular weight of 50–55 kDa was equal in all samples, this protein was used as housekeeping internal control. Moreover, since {gamma}-catenin expression was unchanged in both healthy and {delta}-SG null hamster hearts, it served as further housekeeping internal control. The intensity of specific bands was evaluated using the NIH Pro-Image 1.62 software after blot scanning by Umax Vista Super Speedy scanner. The up- or downregulation of proteins was expressed as percent variation of {delta}-SG null hamsters vs. healthy controls after normalization with the housekeeping internal control.

2.5. Northern blot analysis
Total RNA was extracted from hamster hearts using Trizol (Invitrogen, Milan, Italy) and 10 µg/samples were size-fractionated in 1.2% agarose gel containing 2.2 M fomaldehyde and transferred to Hybond N membrane (Amersham Pharmacia Biotech., Buckinghamshire, UK) using 20 x SSC (1 x SSC=0.15 M NaCl/0.015 M sodium citrate). RNA was covalently bound by UV irradiation and filters were hybridized for 16 h at 65 °C with [{alpha}-32P]dCTP-labelled GAPDH, β-catenin and Wnt-5a cDNA probes in MiracleHyb hybridization solution (Stratagene Europe, Amsterdam, NL). After hybridization, filters were washed twice for 15 min in 2 x SSC/0.1% SDS at room temperature and once with 0.1 x SSC/0.1% SDS for 30 min at 60 °C and exposed to Biomax MS film (Eastman Kodak, Rochester, NY) at –70 °C.

GAPDH, β-catenin and Wnt-5a were PCR products obtained from RNA of healthy and {delta}-SG null hamster hearts using 1 µg of total RNA, 200 U of Superscript II Reverse Transcriptase (Invitrogen), 10 mM DTT, 20 U of RNaseOut (Invitrogen), 2.5 µM of random hexamers, 1 mM of each dNTP and 1 x buffer provided by the manufacturer in 20 µl (final volume). The reaction was performed at 42 °C for 60 min. The cDNA product (1 µl) was amplified with 1 U of Platinum Taq DNA polymerase (Invitrogen) in a final volume of 50 µl containing 200 µM of each dNTP and 50 pmol of each primer. The sequences of the GAPDH primers were as follows: 5'-TCTACTGGTGCTTTCACCACCATG-3' forward and 5'-CTGCTTCACCACCTTCTTGATGTC-3' reverse (sequence data available from EMBL/GeneBank under accession number M32599 [GenBank] ). The sequences of the β-catenin primers were as follows: 5'-CCTGAGACGCTAGATGAGGCATGC 3' forward and 5' CTTTCACCACTCTGCTTGTGGTCC-3' reverse (accession number M90364 [GenBank] ). The sequences of Wnt-5a primers were as follows: 5'-CCCTCGCCATGAAGAAGCCCATTG-3' forward and 5'-CCACCACCAGTCCCGAGGCAGGTC-3' reverse (accession number X56842 [GenBank] ). Amplification cycles consisted of 94 °C for 1 min, 65 °C for 1 min and 72 °C for 1 min, preceded by the first step at 94 °C for 2 min to allow the enzyme activation.

Primers (Invitrogen) were designed to span on different exons so as not to amplify contaminating genomic DNA. To confirm the identity of the amplicons, PCR products were hybridized with an internal oligonucleotide labelled with DIG oligonucleotide 3'-end labelling kit (Roche Diagnostic, Mannheim, Germany) according to the instructions of the manufacturer. The intensity of the specific band was evaluated using NIH Pro-Image 1.5 software after blot scanning by Umax Vista Super Speedy scanner. The up- or downregulation of transcripts was expressed as percent variation of {delta}-SG null hamsters vs. healthy controls after normalization with the housekeeping internal control (GAPDH).

2.6. Immunohistochemistry
{delta}-Sarcoglycan-deficient hamster hearts and age-matched controls were excised and fixed overnight in 10% buffered formalin. After paraffin embedding, each sample was cut into 5 µm sections. Myocardial sections of {delta}-SG null and control animals were analysed for the reactivity with properly diluted polyclonal antibodies against the following antigens: {alpha}, β-, {gamma}-catenin, N-cadherin, APC, Wnt and GSK3β (Santa Cruz). Antigen-antibody complexes were visualised by 3-amino-9-ethylcarbazole (AEC) reaction as previously described [28]. Human heart specimens were visualised by 3,3'-diaminobenzidine (DAB). Sections were counterstained with hematoxylin. Each observation was performed independently three times. For the hamsters, three animals per each age group were analyzed. Staining intensity was semiquantitatively classified as negative (–), weak (+) and strong (++). No reactivity was observed with normal rabbit or goat control serum.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Alteration of cell/cell contacts in the hamster hypertrophic cardiomyopathy
To determine the morphological features of the hamster hypertrophic cardiomyopathy (UM-X7.1 strain), myocardial tissue of {delta}-SG null and control animals was analyzed at different ages by light and transmission electron microscopy.

By light microscopy, no significant morphological alterations were detectable in 30-day-old {delta}-SG null vs. age-matched healthy myocardial tissue (Fig. 1A–B). Conversely, in 90- and 150-day-old cardiomyopathic hamster hearts, the tissue architecture was deeply altered and cells were chaotically oriented (Fig. 1D–F). Cardiomyocytes were often enlarged, binucleated and odd-shaped (Fig. 1D), while extracellular matrix deposition was increased paralleling the myocardial damage severity. In addition, myocardium was patched at random by large necrotic areas (Fig. 1D–F).


Figure 1
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  		Fig. 1 Morphological features of the hamster hypertrophic cardiomyopathy. Light microscopy performed on healthy and cardiomyopathic hamster myocardium. Lack of significant morphological alterations in {delta}-SG null vs. healthy 30-day-old myocardial tissue (A–B). Alterations of tissue architecture in 90- and 150-day-old cardiomyopathic hamster hearts (D–F) compared with age-matched healthy controls (C–E). Semithin sections were stained with tolouidine blue (x 400).

 
The ultrastructural analysis performed on ultrathin sections showed the presence of fully developed and few still immature cardiomyocytes in healthy and cardiomyopathic hearts aged 30 days (data not shown). In 90- and 150-day-old healthy hearts, the intracellular structure and orientation of cardiomyocytes were normally represented (Fig. 2A). Conversely, in {delta}-SG null 90- and 150-day-old hamsters, cardiomyocytes were enlarged and their intracellular organization was severely altered. Sarcomeres were disorganized; myofibrils lost their orientation and were surrounded by vacuoles; Z band intervals were irregular (Fig. 2B). A diffuse alteration of cell/cell contacts was characteristically found in {delta}-SG null hamsters. Fig. 2 shows representatively alterations of intercalated disks sectioned lengthwise in cardiomyopathic (Fig. 2D) compared to healthy hearts (Fig. 2C). Intercalated disks were chaotically located, structurally redundant and swirling (Fig. 2D and E). Furthermore, in cardiomyopathic hearts, basal membrane detachment, very likely due to the joint action of missed {delta}-SG and mechanical stress, was often observed (Fig. 2F).


Figure 2
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  		Fig. 2 Alterations of cell/cell contacts in the hamster hypertrophic cardiomyopathy. Ultrastructural analysis performed on healthy and cardiomyopathic hamster hearts (90 days). Healthy hamster myocardium (A) (x 4900). Sarcomere disorganization in the hamster hypertrophic cardiomyopathy with lack of miofibril orientation (B) (x 4900). Alterations of intercalated disks (cut lengthwise) in the hamster hypertrophic cardiomyopathy (D) (x 14000) compared to healthy heart (C) (x 14000). In cardiomyopathic hearts, intercalated disks were chaotically located and structurally swirling (E) (x 21000). Basal membrane detachment in cardiomyopathic hearts (solid arrowhead) as compared to integral basal membrane (arrow) (F) (x 21000).

 
3.2 Increased expression of N-cadherin and β-catenin in the hamster hypertrophic cardiomyopathy
Morphological analysis evidenced the presence of altered cell/cell contacts in {delta}-SG null hypertrophic cardiomyopathic hearts. In particular, the alteration of intercalated disks in the damaged myocardium suggested the possible deregulation of molecules involved in cell/cell adhesion. Our investigation was directed to evaluate, in {delta}-SG null cardiomyopathic hearts, the expression and organization of the cadherin/catenin complex, a major molecular structure involved in cell/cell contacts.

To compare the expression levels of N-cadherin and catenins in hamster cardiomyopathic hearts vs. age-matched controls, Western blot analysis was performed on myocardium homogenates from three different pools of hamster hearts. A representative experiment is shown in Fig. 3A. The normalization of protein expression was obtained using as housekeeping internal control a protein that was unchanged by Ponceau Red staining in both groups of animals (Fig. 3, Panel A, control). The expression of N-cadherin showed a 42% decrease in 30-day-old cardiomyopathic hamster hearts vs. age-matched controls, while a 50% and 30% increase was observed in 90- and 150-day-old cardiomyopathic hearts, respectively (Fig. 3, Panel B). Conversely, a {alpha}-catenin decrease (–15%) was observed in 30-day-old {delta}-SG null hamsters only. However, a relevant increase in β-catenin expression was found in 90- (62%) and 150 (70%)-day-old cardiomyopathic hamsters vs. controls (Fig. 3, Panel B). To determine whether the apparent increase of β-catenin was due to an incremented protein production, the Northern blot analysis was performed. As showed in Fig. 3, Panel C, the level of β-catenin transcript was similar in cardiomyopathic and healthy hamster hearts. Moreover, the expression of {gamma}-catenin was unchanged in cardiomyopathic vs. healthy animals.


Figure 3
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  		Fig. 3 N-Cadherin and β-catenin in the hamster hypertrophic cardiomyopathy. Western blot showing the reactivity of polyclonal antibodies to N-cadherin and catenins in healthy and cardiomyopathic hearts (Panel A). The intensity of each specific band was expressed as densitometric units (DU) after normalization with the housekeeping internal control (Panel B). Northern blot showing that β-catenin transcript level was similar in healthy and cardiomyopathic hamster hearts (Panel C). The intensity of the specific band was expressed as DU after normalization with the housekeeping internal control (GAPDH).

 
The expression of the cadherin/catenin complex was further analyzed along with its localization by immunohistochemical analysis. Our results demonstrated a different expression of cadherin/catenin complex proteins in cardiomyopathic vs. healthy hamster myocardium (Table 1). N-Cadherin was highly expressed in both intercalated disks and cytosol (++) of 30-day-old healthy hearts, while, in age-matched {delta}-SG null myocardium, it was mostly located in intercalated disks (+). In older animals, N-cadherin was almost exclusively present in intercalated disks, but the signal was noticeably stronger in cardiomyopathic myocardium (++) (Table 1, Fig. 4A–B). In 30-day-old healthy hearts, {delta}-catenin was expressed in both intercalated disks (++) and cytosol (+), but only in the intercalated disks (+/++) of {delta}-SG null myocardia. However, {alpha}-catenin expression remained similar in 90- and 150-day-old cardiomyopathic hamsters vs. age-matched controls.


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  		Table 1 Immunohistochemical expression of the N-cadherin/catenins complex in healthy and hypertrophic cardiomyopathic hamster hearts

 

Figure 4
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  		Fig. 4 β-Catenin accumulation in intercalated disks of hypertrophic cardiomyopathic hamster hearts. Representative immunohistochemical staining for N-cadherin (A–B) and β-catenin (C–D) in 90-day-old healthy (A–C) and cardiomyopathic hearts (B–D). Normal rabbit serum served as negative control (E–F). Immunoperoxidase counterstained with haematoxylin. Original magnification: x 400.

 
The expression of β-catenin was similar in 30-day-old animals (i.e. + in both intercalated disks and cytoplasm). However, the β-catenin signal was remarkably stronger in the hypertrophic myocardium (++) vs. controls (+/–) in both 90- and 150-day-old animals. At these ages, β-catenin was restricted in the intercalated disk of {delta}-SG cardiomyocytes, while no nuclear and cytoplasmic staining was observed (Fig. 4C–D). Conversely, no significant modifications between the normal and cardiomyopathic myocardium were found in {gamma}-catenin expression at all ages (Table 1). No reactivity was observed when non-immune rabbit serum was used as negative control (Fig. 4E–F).

In dilated cardiomyopathic hamster hearts (TO2 strain), the immunohistochemical analysis showed a modest β-catenin accumulation in intercalated disks in respect to healthy controls (data not shown).

3.3. Differential expression of adenomatous polyposis coli gene products, Wnt and GSK3β in the hamster hypertrophic cardiomyopathy
The increased stabilization and subsequent accumulation of β-catenin at the cell/cell adhesion sites suggested investigating the expression of three proteins involved in the regulation of β-catenin stability: the APC, Wnt and GSK3β.

Immunohistochemical and Western blot analyses using two different antibodies recognizing the last 19 C-terminal and 20 N-terminal amino acids, respectively, evaluated APC protein expression. Combining the two antibodies, it is possible to detect 16 different APC isoforms differentially expressed in various diseases [29]. Immunohistochemical analysis did not show any significant difference in APC expression and localization in 30-day-old healthy vs. cardiomyopathic hearts. Conversely, in 90- and 150-day-old healthy hearts, APC was finely dispersed in the cytosol (Fig. 5, Panel A, a), while, in cardiomyopathic hearts, it was mostly present in intercalated disks (Fig. 5, Panel A, b). Western blot analysis was then performed in 90-day-old animal hearts and it demonstrated the presence in all myocardium samples of different APC isoforms with an apparent molecular weight ranging between 30 and 300 kDa. However, in the cardiomyopathic vs. healthy hamster myocardium, the 60-kDa APC isoform was increased, while the 45-kDa isoform was decreased, similarly to human hypertrophic hearts [29]. In addition, the isoforms with MW of 150, 200 and 290 kDa, specifically expressed in postmitotic cells [29], were decreased in {delta}-SG null hamster hearts (Fig. 5, Panel B).


Figure 5
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  		Fig. 5 Different expression of the adenomatous polyposis coli gene products in the hamster hypertrophic cardiomyopathy. Immunohistochemical localization of APC products in 90-day-old healthy (Panel A, a) and cardiomyopathic hearts (Panel A, b). Immunoperoxidase counterstained with haematoxylin. Original magnification: x 400. Western blot analysis showing the different expression of APC isoforms (asterisks and arrows) in healthy (Panel B, lane 1) and cardiomyopathic hearts (Panel B, lane 2).

 
Immunohistochemical analysis of Wnt and GSK3β demonstrated that both proteins were expressed in the cytosol (+ and ++, respectively) of healthy hamster hearts independently of age (Table 1) (Fig. 6, Panels A, a and C, c, respectively). In hypertrophic cardiomyopathic hearts, Wnt expression was increased and localized peripherally in the plasmamembrane of cardiomyocytes (++) (Table 1) (Fig. 6, Panel A, b). On the other hand, the level of GSK3β diminished in cardiomyopathic hearts of 90- and 150-day-old {delta}-SG null hamsters (+) compared with healthy hamsters (++) (Table 1) (Fig. 6, Panel C, d and c, respectively).


Figure 6
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  		Fig. 6 Increased expression of Wnt paralleling a decreased expression of GSK3β in the hamster hypertrophic cardiomyopathy. Representative immunohistochemical staining for Wnt in healthy (Panel A, a) and cardiomyopathic hearts (Panel A, b). Immunoperoxidase counterstained with haematoxylin. Original magnification: x 400. Northern blot showing a twice increase in Wnt 5a transcript in cardiomyopathic hearts (hy) vs. controls (ctr). The intensity of the specific band was expressed as DU after normalization with the housekeeping internal control (GAPDH) (Panel B). Representative immunohistochemical staining for GSK3β in healthy (Panel C, c) and cardiomyopathic hearts (Panel C, d). Immunoperoxidase counterstained with haematoxylin. Original magnification: x 400.

 
To corroborate Wnt upregulation, Northern blot analysis was performed using a probe recognizing Wnt-5a that is expressed at high levels in mouse hearts [30]. As shown in Fig. 6, Panel B, Wnt transcript increased twice in 90-day-old cardiomyopathic vs. normal myocardium indicating that the upregulation of Wnt maintains elevated levels of β-catenin in {delta}-SG null cardiomyocytes.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The present investigation demonstrated that, in {delta}-SG null hamster hearts, the cardiomyocyte/basal membrane junction is destroyed and intercalated disks are chaotically located, structurally redundant and swirling. The alteration of intercalated disks in the damaged myocardium suggested the possible deregulation of molecules involved in cell/cell adhesion. In fact, in hypertrophic cardiomyopathic cardiomyocytes, we observed for the first time remarkably increased levels of N-cadherin and β-catenin that were mainly located in intercalated disks. Such an accumulation of β catenin in intercalated disks was determined by a post-translational mechanism; in fact, the level of β-catenin transcript was unchanged in cardiomyopathic vs. healthy hamster hearts, while significant modifications were present in downstream protein systems regulating cadherin/catenin expression and function. In physiological conditions, the level of β-catenin is regulated by Wnt signaling that stabilizes cytosolic β-catenin. In the absence of Wnt signals, β-catenin is phosphorylated by GSK3β, ubiquinated and degraded in proteasome [23,31]. In cardiomyopathic hearts, an increased expression of Wnt associated with a simultaneous decrease in GSK3β expression. Consequently, β-catenin was no more phosphorylated and degraded and was made available to be translocated to plasma membrane or nucleus [23]. The accumulation of β-catenin in intercalated disks of {delta}-SG null cardiomyocytes indicates that, in hypertrophic cardiomyopathic hearts, this molecule is mainly implicated in mediating cell/cell adhesion rather than transcription of Wnt target genes [23,30]. APC proteins that, in our animal model, are particularly abundant in intercalated disks might mediate the β-catenin accumulation. It has been indicated that APC concur to concentrate and deliver β-catenin to the plasma membrane [32,33]. The "taxi function" of APC is, very likely, played by specific APC isoforms. In this respect, we observed that the 60 kDa APC isoform is particularly abundant in hypertrophic cardiomyopathic hamster hearts.

The observation that the {delta}-SG null hamster cardiomyopathy can alternatively be hypertrophic or dilated and the results of recent investigations in cardiomyopathic patients conclusively demonstrated that a single mutation could generate a spectrum of cardiomyopathic phenotypes. The interplay of mutations with other concurrent genetic and/or environmental factors and the activation of aberrant intracellular signaling pathways [34,35], very likely, cause the protean clinical outcome of cardiomyopathies. The {delta}-SG null hamster represents a clear example of this phenomenon. In fact, the pathogenic mechanism of cardiomyopathy might result from the integrated effects induced by the lack of sarcoglycan–sarcospan complex in vascular smooth muscle with consequent functional coronary alterations [11,12], and, among others, the occurrence of an additional mitochondrial mutation in hypertrophic, but not dilated cardiomyopathic animals [36]. However, the perturbation of sarcolemmal integrity caused by the lack of {delta}-SG expression suggests, besides changes in the sarcoglycan–sarcospan complex in vascular smooth muscle [11,12], a possible cell/cell contact alteration, very likely, caused by the increased local mechanical stress. The cell/cell adhesion perturbation activates several aberrant intracellular mechanisms contributing to cardiomyocyte damage. Among others, cadherins and catenins constitute a complex whose integrity is necessary for cell/cell adhesion [37]. N-Cadherin and the catenins are homogeneously distributed in the peripheral region of healthy cardiomyocytes during fetal life, while they are localized in intercalated disks during adulthood [38]. In neonatal hearts, Wnts play a role in the organ morphogenesis stimulating fibroblast-dependent cell aggregation and inducing an augmented expression of N-cadherin [30]. The Wnt signaling role in myocardium remodeling has also been confirmed in hypertrophic and ischemic rat hearts where the expression of the Wnt receptor Frizzled was significantly increased [39]. Recently, it has also been proved that different APC isoforms are differently expressed in hypertrophic vs. healthy hearts suggesting an isoform specific role in cardiac remodeling [29]. Interestingly, a remarkable cadherin/catenin system dysregulation occurs in hypertrophic cardiomyopathic hamsters, while in dilated cardiomyopathic hamsters a slight increase in the β-catenin signal is detectable in intercalated disks. Hypertrophic cardiomyopathic hamsters suffer for an additional mitochondrial mutation in respect to the equivalent dilated model [36], but the causative link between the mitochondrial mutation and the increased expression of β-catenin remains to be elucidated.

Taken together, our results demonstrate that hamster cardiomyocytes are able to reorganize cell/cell contacts in order to compensate for the looser cell/extracellular matrix adhesion caused by the lack of {delta}-SG. The reorganization of cell/cell adhesion is mainly contributed by the cadherin/catenin system, which is differently regulated to sustain cell structural rather than signaling needs. This process causes relevant consequences in the determination of the cardiomyocyte phenotype and clinical outcome. In fact, the segregation of β-catenin in intercalated disks implies that cytoplasmic β-catenin level is lowered and nuclei are deprived of stimuli needed to maintain a physiological gene activity. Accordingly, the present study demonstrated the absence of β-catenin specific signal in the cytoplasm and nuclei of {delta}-SG null cardiomyocytes, while previous observations showed an altered transcription factor pattern [40] and continued activation of the embryonic gene program [41] in adult hamster cardiomyopathic hearts. On the other hand, the accumulation of β-catenin in intercalated disks could play a role in determining the previously described plasmalemma rigidification in {delta}-SG null cardiomyocytes [42]. This mechanism could contribute to increased myocardial wall stiffness and left ventricular end-diastolic pressure (LVEDP) as well as to modify intercellular electric impedance in hypertrophic cardiomyopathic hamsters [41] and men [25]. Consistently with {delta}-SG null hamster hearts, our results in human hypertrophic cardiomyopathy demonstrated that a remarkable accumulation of N-cadherin and β-catenin occurs in enlarged and disorganized intercalated disks (Fig. 7B–D). The similar modifications of the cadherin/catenin complex found in hamster and human hypertrophic cardiomyopathy suggest that the alteration of the β-catenin pathway, very likely, plays a fundamental role in the modulation of genes directly related to the onset of the hypertrophic phenotype.


Figure 7
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  		Fig. 7 β-Catenin accumulation in intercalated disks of hypertrophic cardiomyopathic human heart. Immunohistochemical staining for N-cadherin (A–B) and β-catenin (C–D) in healthy (A–C) and cardiomyopathic human heart (B–D). Immunoperoxidase counterstained with haematoxylin. Original magnification: x 400.

 


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The study has been supported by grants from Cofin 2002, ASI 2000 and Telethon N. 1126. Authors wish to thank Syntech, Rome, for technical and scientific support.


   Notes
 
Time for primary review 26 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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